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D Smith

Publications and source records attributed to D Smith.

At least 433 records · Page 24Linked to original sources
PubMed1997

Delineation of the arginine- and tetrahydrobiopterin-binding sites of neuronal nitric oxide synthase.

Nitric oxide synthase (EC 1.14.13.39) catalyses the conversion of arginine, NADPH and oxygen to nitric oxide and citrulline, using haem, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin), calmodulin, FAD and FMN as cofactors. The enzyme consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region that contains the arginine and tetrahydrobiopterin sites and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. By using domain boundaries defined by limited proteolysis of full-length enzyme, recombinant haem-binding regions of rat brain neuronal nitric oxide synthase were expressed and purified. Two proteins were made in high yield: one, corresponding to residues 221-724, contained bound haem and tetrahydrobiopterin and was able to bind Nomega-nitro-l-arginine (nitroarginine) or arginine; the other, containing residues 350-724, contained bound haem but was unable to bind tetrahydrobiopterin, nitroarginine or arginine. These results showed that rat brain neuronal nitric oxide synthase contains a critical determinant for arginine/tetrahydrobiopterin binding between residues 221 and 350. Limited proteolysis with chymotrypsin of the former protein resulted in a new species with an N-terminal residue 275 that retained the ability to bind nitroarginine, further defining the critical region for arginine binding as being between 275 and 350. Comparison of the sequences of nitric oxide synthase and the tetrahydrobiopterin-requiring amino acid hydroxylases revealed a similarity in the region between residues 470 and 600, suggesting that this might represent the core region of the pterin-binding site. The stoichiometries of binding of substrate and cofactors to the recombinant domains were not more than 0.5 mol/mol of monomer, suggesting that there might be a single high-affinity site per dimer.

Amino Acid Sequence↗
PubMed1997

Highly potent irreversible inhibitors of neutrophil elastase generated by selection from a randomized DNA-valine phosphonate library.

We incorporated a phosphonate irreversible inhibitor of neutrophil elastase into a randomized DNA library and, using the SELEX process, iteratively selected these assemblies for the most potent elastase inhibitors. The inhibitors were selected against purified elastase and against secreted elastase in the presence of activated neutrophils. Very active aptamer inhibitors were obtained by both methods, with second-order rate constants for inactivation of human neutrophil elastase ranging (1-3) x 10(8) M(-1) min(-1). These rates exceed those of any reported irreversible inhibitor of elastase and exceed the previous best phosphonate inhibitors by 80-fold. The selected inhibitors are also significantly more potent than alpha-1 proteinase inhibitor in blocking degradation of elastin by activated neutrophils. In contrast to a previous experiment [Smith et al. (1995) Chem. Biol. 2, 741-750], a single-enantiomer form of the valyl phosphonate was used rather than a racemic mixture. Our analysis shows that this use of a chirally resolved valyl phosphonate results in selection of much more potent inhibitors and that these inhibitors specifically potentiate a single enantiomeric form of the phosphonate.

Cathepsin G↗
PubMed1997

Tacrolimus rescue therapy for renal allograft rejection--five-year experience.

Over the 5 year period from 7/14/1989 until 5/24/1994, we have attempted graft salvage with tacrolimus conversion in a total of 169 patients (median age 33 years, range 2-75 years) with ongoing rejection on baseline CsA immunosuppression after failure of high dose corticosteroids and/or antilymphocyte preparations to reverse rejection. The indications for conversion to tacrolimus were ongoing, biopsy confirmed rejection in all patients. The median interval to tacrolimus conversion was 2 months (range 2 days to 55 months; mean 4.3+/-2.6 months) after transplantation. All patients had failed high dose corticosteroid therapy and 144 (85%) of the 169 patients had received at least one course of an antilymphocyte preparation plus high dose corticosteroid therapy prior to conversion. Twenty-eight patients (17%) were dialysis-dependent at the time of conversion owing to the severity of rejection. With a mean follow-up of 30.0+/-2.4 months (median 36.5 months, range 12-62 months), 125 of 169 patients (74%) have been successfully rescued and still have functioning grafts with a mean serum creatinine (SCR) of 2.3+/-1.1 mg/dl. Of the 144 patients previously treated with antilymphocyte preparations, 117 (81%) were salvaged. Of the 28 patients on dialysis at the time of conversion to tacrolimus, 13 (46%) continue to have functioning grafts (mean SCR 2.15+/-0.37 mg/dl) at a mean follow-up of 37.3+/-16.7 months. In the 125 patients salvaged, prednisone doses have been lowered from 28.0+/-9.0 mg/d (median 32, range 4-60 mg/d) preconversion to 8.5+/-4.1 mg/d (median 12 mg/d, range 2.5-20 mg/d) postconversion. Twenty-eight patients (22.4%) are currently receiving no steroids. This 5 year experience demonstrates that tacrolimus has sustained efficacy as a rescue agent for ongoing renal allograft rejection. Based on these data, we recommend that tacrolimus be used as an alternative to the conventional drugs used for antirejection therapy in renal transplantation.

Adolescent↗
PubMed1997

Smoking, nicotine dependence, and depressive symptoms in the CARDIA Study. Effects of educational status.

The present study was designed to determine whether depressive symptoms are independently associated with smoking and nicotine dependence among cigarette smokers, using 1990-1991 data from the Coronary Artery Risk Development in Young Adults (CARDIA) Study. A total of 3,933 participants (788 black men, 1,090 black women, 974 white men, and 1,081 white women) aged 23-35 years were included. Analyses were stratified by race and sex. Depressive symptoms were measured by means of the Center for Epidemiologic Studies Depression (CES-D) Scale. Nicotine dependence was defined as smoking one's first cigarette of the day within 30 minutes of awakening. Analysis of covariance was used to control for potential covariates (age, body mass index, alcohol consumption, and education). In unadjusted comparisons, smokers had more depressive symptoms than never smokers in all groups except white men; this relation showed little change after adjustment for age, body mass index, and alcohol consumption. However, after adjustment for education in addition to the above variables, these differences became attenuated and were significant only among white women (adjusted CES-D score difference = 1.9, p < 0.02). When analyses were further stratified by nicotine dependence, dependent smokers had higher CES-D scores than never smokers in all groups. The differences again became attenuated when education was added to the model, and were significant only among black women (adjusted CES-D score difference = 2.3, p < 0.01). These results indicate that although smoking in general and nicotine-dependent smoking in particular are related to symptoms of depression, controlling for educational level attenuates these relations.

Adult↗
PubMed1997

High-frequency unilateral thalamic stimulation in the treatment of essential and parkinsonian tremor.

Pharmacologic treatment for essential tremor and the tremor of Parkinson's disease is often inadequate. Stereotaxic surgery, such as thalamotomy, can effectively reduce tremors. We performed a multicenter trial of unilateral high-frequency stimulation of the ventral intermedius nucleus of the thalamus in 29 patients with essential tremor and 24 patients with Parkinson's disease, using a blinded assessment at 3 months after surgery to compare clinical rating of tremor with stimulation ON with stimulation OFF and baseline and a 1-year follow-up. Six patients were not implanted because of lack of intraoperative tremor suppression (2 patients), hemorrhage (2 patients), withdrawal of consent (1 patient), and persistent microthalamotomy effect (1 patient). A significant reduction in both essential and parkinsonian tremor occurred contralaterally with stimulation. Patients reported a significant reduction in disability. Measures of function were significantly improved in patients with essential tremor. Complications related to surgery in implanted patients were few. Stimulation was commonly associated with transient paresthesias. Other adverse effects were mild and well tolerated. Efficacy was not reduced at 1 year. Chronic high-frequency stimulation is safe and highly effective in ameliorating essential and parkinsonian tremor.

Aged↗
PubMed1997

Neutrophil-mediated damage to vascular endothelium in the spontaneously hypertensive rat.

Lysis of aortic endothelial cells (EC) by neutrophils from spontaneously hypertensive rats (SHR) was investigated using a nonradioactive cytotoxicity assay. Interleukin-1-activated EC, but not unstimulated EC, were effective target cells for lysis by SHR neutrophils. Supernatants from activated neutrophil did not exert a cytotoxic effect on EC. Inhibitors of reactive oxygen species did not affect the cytotoxicity of neutrophils on EC. In contrast, inhibitors of serine protease and elastase markedly inhibited the cytotoxicity of neutrophils on EC. Antibodies against the endothelial cell surface ligands ICAM-1 (CD54) and E-selectin (CD62E) inhibited the adhesion and cytotoxicity of activated neutrophils on EC. The cytotoxicity of neutrophils required direct cell-to-cell contact because separating them with a microporous membrane abrogated the neutrophil-mediated cytotoxic activity. These results demonstrate that SHR neutrophils possess potent cytotoxicity against cytokine-activated EC. Neutrophil-mediated damage of EC could contribute to organ damage in hypertension under conditions of local or systemic activation of neutrophils.

Animals↗
PubMed1997

Cryosurgery of dunning AT-1 rat prostate tumor: thermal, biophysical, and viability response at the cellular and tissue level.

This study investigates cryodestruction of the Dunning AT-1 rat prostate tumor at the single cell, tissue slice, and in vivo levels. The thermal history around a 3-mm-diameter cylindrical cryosurgical probe was predicted by solving the bioheat equation in a one-dimensional cylindrical geometry. At various radial positions in the iceball this thermal history was approximated by a constant cooling rate and a final, steady-state temperature (or end-temperature). The predicted cooling rates and end temperatures ranged from > or = 1000 degrees C/min to 5 degrees C/min and -196 degrees C to -20 degrees C, respectively. These cooling rates and end-temperatures were then imposed on single AT-1 cells, AT-1 tissue slices in vitro and AT-1 tumors in vivo. The single cells and tissue slices were frozen by LN2 immersion, copper block slam-freezing, or controlled cooling on a cryomicroscope or a directional solidification stage. LN2 immersion is lethal to AT-1 cells (presumably due to intracellular ice formation), while cooling at 5-100 degrees C/min leaves some viable cells (at end-temperatures ranging between -20 and -40 degrees C). AT-1 tumor slices show extensive intracellular ice formation due to slam cooling, extensive dehydration at 100 degrees C/min, and total dehydration at rates < or = 10 degrees C/min to end temperatures below -10 degrees C. Postfreeze culture and histology of the AT-1 tissue show that extensive intracellular ice formation is lethal, while cellular dehydration and vascular engorgement leave viable cells (at end-temperatures between -20 and -40 degrees C). Based solely on the single cell and in vitro tissue damage achieved by cooling rates and end-temperatures, a sizable portion of a cryosurgically frozen tumor would be expected to survive. However, in vivo cryosurgery performed on AT-1 tumors demonstrated that the tissue was damaged throughout the cryolesion, even at the periphery where the thermal history would be expected to allow single cells and tissue slices to survive in vitro. Taken together, these results suggest that damage mechanisms other than those due to cooling rate and end-temperature may be responsible for the increased cellular destruction at the periphery of the iceball in vivo and that cooling rate is less important than end-temperature in determining cryosurgical damage in AT-1 tumors. Experiments are ongoing to determine if the time held at an end temperature, thawing rate, vascular response, or other mechanisms are primarily responsible for the enhanced destructive capability in vivo.

Animals↗
PubMed1997

Magnesium deficiency increases ketamine sensitivity in rats.

PURPOSE: Inhibition of the NMDA receptor likely contributes to ketamine's neurodepressive properties. Magnesium also inhibits the NMDA receptor by binding to a site associated with the ketamine-binding domain. Electrophysiological studies suggest that magnesium prevents ketamine from binding to the NMDA receptor and thereby prevents ketamine inhibition. We undertook an in vivo study to determine if magnesium deficiency was associated with an increased sensitivity to ketamine. METHODS: Weanling rats were maintained on a Mg(2+)-deficient or control diet for 14 days. In Study I, rats were anaesthetized then sacrificed and the Mg2+ concentrations in the brain and plasma were measured. In a second prospective study, experimental animals were rendered hypomagnesaemic and the potency of 125 mg.kg-1 ip ketamine was evaluated. Animals were then were fed a Mg(2+)-containing diet and ketamine sensitivity was re-examined 14 days later. RESULTS: The Mg(2+)-deficient diets rendered the rats hypomagnesaemic as indicated by the brain and plasma concentration of Mg2+. In Study 2, the time to loss of righting reflex was shorter; 1.9 +/- 0.3 min (n = 12) and 2.6 +/- 0.2 min (n = 16, P < 0.05), whereas the latency to toe pinch was prolonged: 25.0 +/- 5.8 min (n = 12) vs 3.1 +/- 2.1 min (n = 16, P < 0.05) in the Mg(2+)-deficient compared with age-matched control animals, respectively. The hypomagnesaemic animals had a higher death rate following ketamine injection. The increased sensitivity to ketamine was no longer apparent when the animals were re-tested following replenishment of Mg2+. CONCLUSION: Hypomagnesaemia is associated with an increased sensitivity to ketamine.

Anesthetics, Dissociative↗
PubMed1997

Plasma diamine oxidase activities in renal dialysis patients, a human with spontaneous copper deficiency and marginally copper deficient rats.

OBJECTIVES: Intestine and kidney are generally the most concentrated sources of the copper metalloenzyme diamine oxidase (DAO). Clinically, plasma DAO activities are used to diagnose disruptions in intestinal integrity. This study determined whether DAO activities were also affected by kidney injury or copper nutritional status. DESIGN AND METHODS: Plasma DAO activities were measured in renal dialysis patients without diagnosed intestinal disease (n = 75), controls (n = 23), an adult with spontaneous copper deficiency before and after copper repletions, and in rats fed either adequate or marginal copper diets (8 or 2 mg copper/kg diet) for 7 months. RESULTS: This study found high DAO activities in renal dialysis patients and low activities during spontaneous copper deficiency. Low activities were also seen for marginally copper deficient rats. CONCLUSIONS: Tissue injury-induced elevation of DAO activities is not limited to intestinal injury, and low DAO values may be useful for assessing copper nutritional status.

Adult↗
PubMed1997

Metabolism of rifabutin in human enterocyte and liver microsomes: kinetic parameters, identification of enzyme systems, and drug interactions with macrolides and antifungal agents.

Biotransformation of rifabutin, an antibiotic used for treatment of tuberculosis in patients infected with the human immunodeficiency virus (HIV), and its interactions with some macrolide and antifungal agents were studied in human intestinal and liver microsomes. Both liver and enterocyte microsomes metabolized rifabutin to 25-O-deacetylrifabutin, 27-O-demethylrifabutin, and 20-, 31-, and 32-hydroxyrifabutin. The same products (except 25-O-deacetylrifabutin) were formed by microsomes from lymphoblastoid cells that contained expressed CYP3A4. The apparent Michaelis-Menten constant (Km); approximately 10 to 12 mumol/L) and maximal velocity (Vmax; approximately 100 pmol/min/mg of protein) values for CYP-mediated metabolism were similar in liver and enterocyte microsomes. Deacetylation of rifabutin (Km approximately 16 to 20 mumol/L and Vmax approximately 50 to 100 pmol/min/mg of protein) was catalyzed by microsomal cholinesterase. Clarithromycin, ketoconazole, and fluconazole inhibited CYP-mediated metabolism of rifabutin in enterocyte microsomes equally or more potently than in liver microsomes but had no effect on cholinesterase activity. Azithromycin did not inhibit in vitro metabolism of rifabutin. This study provides evidence that CYP3A4 and cholinesterase are major enzymes that biotransform rifabutin in humans and that intestinal CYP3A4 contributes significantly to rifabutin presystemic first-pass metabolism and drug interactions with macrolide and antifungal agents.

Acetylation↗
PubMed1997

The predictive value of race as a clinical prognostic factor among patients with clinically localized prostate cancer: a multivariate analysis of positive surgical margins.

OBJECTIVES: Several investigators have reported that African-American men with clinically localized prostate cancer have poorer survival than do white men. In addition, prostate cancer in African-American men is commonly diagnosed at a more advanced stage of disease. Is race or ethnicity predictive of outcome of clinically localized prostate cancer? It has been reported that the presence of positive surgical margins significantly influences time to progression independently of other prognostic factors. Therefore, we have elected to conduct a multivariate analysis of clinical factors including race as potential predictors of positive surgical margin outcome. METHODS: We studied 369 consecutive men (120 African-American and 249 white) who had radical prostatectomies at a single institution. Comparisons by race of Gleason score, stage, presence of positive surgical margins, and mean preoperative prostate-specific antigen (PSA) level were carried out. RESULTS: Our data demonstrate that African-American men have more pathologically locally advanced prostate cancer than do white American men: 69% among blacks compared with 57% among whites. However, the difference in rate of positive surgical margins between blacks and whites is statistically significant: 58% among blacks versus 40% among whites (P = 0.002). Four factors were predictive of positive surgical margins: preoperative PSA level, race, clinical stage, and Gleason score. CONCLUSIONS: We have demonstrated that race is an independent predictor of positive surgical margins among patients with clinically localized prostate cancer and should be included in treatment decisions. In addition, the risk of positive surgical margins increases noticeably when PSA is greater than 10 ng/mL.

Aged↗
PubMed1997

The expression of voltage-dependent calcium channel beta subunits in human cerebellum.

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.

Aged↗
PubMed1997

Forces produced by lip bumpers on mandibular molars.

The purpose of this study was to measure the forces produced by a lip bumper on the mandibular permanent first molars. The forces in a sample of 38 patients were measured bilaterally with specially designed gauges at rest but with their lips lightly touching, speaking the words church, phone, and pop, and swallowing water. Forces were compared between two types of lip bumpers, i.e., wire or shield, and between various anteroposterior and vertical positions of the lip bumper. The resting forces produced by the wire lip bumper 2 mm anterior to the incisors and vertically positioned at the middle of the incisor crown were 5.93 +/- 4.84 gm for the left side and 4.66 +/- 4.8 gm for the right. The forces were found to be significantly higher when the wire lip bumper was placed 4 mm anterior to the incisors and at a more gingival position, measuring 16.68 +/- 8.7 gm for the left side and 13.88 +/- 8.28 gm for the right. The shield lip bumper had higher forces both at the center of the incisor as well as when it was positioned gingivally. A large individual variation was observed. There were no statistically significant differences in force levels between male and female subjects. Speaking the words church, pop, and phone, produced forces between 11 and 23 gm, using a wire lip bumper. Swallowing produced the highest forces, between 32 and 36 gm. Lip thickness and height did not appear to affect the force levels.

Acrylic Resins↗
PubMed1997

The effect of chronic lithium treatment on the calibre of axons and nerve fibres in the rat sural nerve.

Cases of peripheral neuropathy have been reported in humans receiving lithium therapy. However, no previous studies have addressed the question of whether chronic lithium treatment causes morphological changes in the peripheral nervous system in experimental animals. The present study used stereological methods to determine whether long-term administration of lithium affected the calibre of the axons or of the nerve fibres in the rat sural nerve. Twenty-two rats were divided into 2 groups and given either no treatment or lithium, with serum levels averaging from 0.5 to 0.7 mmol/l. After 30 weeks of treatment, the animals were killed and the sural nerve was isolated at the level of the knee and was removed. The cross-sectional area of axons and of nerve fibres was estimated by point counting. Compared with the controls, a strong tendency towards a reduced nerve fibre area in the lithium-treated animals was found, with a between-group difference of 1.79 microns 2 (P = 0.06). For the axon area, the difference was 0.73 micron 2 (P = 0.20).

Animals↗
PubMed1997

In vivo imaging of inflammation using an aptamer inhibitor of human neutrophil elastase.

BACKGROUND: We previously reported the isolation of aptamer irreversible inhibitors of human neutrophil elastase. We now report on the application of aptamer technology to the field of diagnostic imaging. RESULTS: The enzyme elastase has been reported to bind to the surface of activated neutrophils. Using a fluorescent flow cytometry assay, we showed that an aptamer inhibitor of elastase also binds preferentially to activated neutrophils. We then tested the ability of the aptamer to image inflammation in vivo in a rat reverse passive Arthus reaction model. The aptamer achieved a peak target-to-background (T/B) ratio of 4.3 +/- 0.6 in 2 hours. IgG, which is used clinically to image inflammation, took a longer time to achieve a lower T/B: 3.1 +/- 0.1 at 3 hours. The difference in T/B values is due to the faster clearance of the aptamer signal from the blood pool. CONCLUSIONS: It is feasible to apply aptamer ligands for use in diagnostic imaging, where they may offer significant advantages over monoclonal antibodies and other reagents.

Animals↗
PubMed1997

Preparation and purification of antibodies specific to human neuronal voltage-dependent calcium channel subunits.

Neuronal voltage-dependent calcium channels (VDCCs) each comprising of alpha 1, alpha 2 delta, and beta subunits, are one mechanism by which excitable cells regulate the flux of calcium ions across the cell membrane following depolarisation Studies have shown the expression of several alpha 1 and beta subtypes within neuronal tissue. The comparative distribution of these in normal human brain is largely unknown. The aim of this work is to prepare antibodies directed specifically to selected subunits of human neuronal VDCCs for use in biochemical and mapping studies of calcium channel subtypes in the brain. Previous studies have defined DNA sequences specific for each subunit Comparison of these sequences allows the selection of unique amino acid sequences for use as immunogens which are prepared as glutathione-S-transferase (GST) fusion proteins in E. coli. Polyclonal antibodies raised against these fusion proteins are purified by Protein A chromatography, followed by immunoaffinity chromatography and extensive adsorptions using the appropriate fusion protein-GST Sepharose 4B columns. The resultant antibodies are analysed for specificity against the fusion proteins by ELISA, and by immunofluorescence and Western immunoblot analysis of recombinant HEK293 cells stably transfected with cDNAs encoding alpha 1, alpha 2 delta and beta subunits.

Animals↗