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Biomedical subjects

D M Brown

Publications and source records attributed to D M Brown.

At least 73 records · Page 4Linked to original sources
PubMed1997

Free radical activity of PM10: iron-mediated generation of hydroxyl radicals.

The purpose of this study was to test the hypothesis that particulate matter < or = 10 microns in aerodynamic diameter (PM10) particles have the ability to generate free radical activity at their surface. We collected PM10 filters from the Edinburgh, United Kingdom, Enhanced Urban Network sampling site, removed particles from the filter, and tested their ability to cause free radical damage to supercoiled plasmid DNA. We found that the PM10 particles did cause damage to the DNA that was mediated by hydroxyl radicals, as shown by inhibition of the injury with mannitol. The PM10-associated hydroxyl radical activity was confirmed using a high-performance liquid chromatography-based assay to measure the hydroxyl radical adduct of salicylic acid. Desferrioxamine abolished the hydroxyl radical-mediated injury, which suggests that iron was involved. Analysis of PM10 filters confirmed the presence of large amounts of iron and leaching studies confirmed that the PM10 samples could release substantial amounts of Fe(III) and lesser amounts of Fe(II). To investigate the size of the particles involved in the hydroxyl radical injury, we centrifuged the suspension of PM10 to clarity, tested the clear supernatant, and found that it had all of the suspension activity. We conclude, therefore, that the free radical activity is derived either from a fraction that is not centrifugeable on a bench centrifuge, or that the radical generating system is released into solution.

Air Pollutants↗
PubMed1997

Free radical activity of industrial fibers: role of iron in oxidative stress and activation of transcription factors.

We studied asbestos, vitreous fiber (MMVF10), and refractory ceramic fiber (RCF1) from the Thermal Insulation Manufacturers' Association fiber repository regarding the following: free radical damage to plasmid DNA, iron release, ability to deplete glutathione (GSH), and activate redox-sensitive transcription factors in macrophages. Asbestos had much more free radical activity than any of the man-made vitreous fibers. More Fe3+ was released than Fe2+ and more of both was released at pH 4.5 than at pH 7.2. Release of iron from the different fibers was generally not a good correlate of ability to cause free radical injury to the plasmid DNA. All fiber types caused some degree of oxidative stress, as revealed by depletion of intracellular GSH. Amosite asbestos upregulated nuclear binding of activator protein 1 transcription factor to a greater level than MMVF10 and RCF1; long-fiber amosite was the only fiber to enhance activation of the transcription factor nuclear factor kappa B (NF kappa B). The use of cysteine methyl ester and buthionine sulfoximine to modulate GSH suggested that GSH homeostasis was important in leading to activation of transcription factors. We conclude that the intrinsic free radical activity is the major determinant of transcription factor activation and therefore gene expression in alveolar macrophages. Although this was not related to iron release or ability to deplete macrophage GSH at 4 hr, GSH does play a role in activation of NF kappa B.

Animals↗
PubMed1997

Osteochondral allografts with an intramedullary muscle flap in rabbits.

A model for hemijoint reconstruction using partially demineralized and lyophilized osteochondral allografts combined with an intramedullary muscle flap is described. The proximal 2/3 of the humerus was resected in 10 rabbits. The remaining defect was reconstructed with either a control lyophilized osteochondral allograft or a lyophilized allograft with a muscle flap filling the marrow cavity. Graft healing was followed by serial radiographs and magnetic resonance imaging. The grafts were harvested at 5 weeks for histologic analysis. By 5 weeks, 4 of 5 control allografts had fractured. In contrast, only 1 allograft with an intramedullary muscle flap showed evidence of a cortical break. Magnetic resonance imaging of control allografts showed a persistent large dead space within the marrow cavity and callus formation only at the outer cortical surface. Magnetic resonance imaging of allografts with an intramedullary muscle flap showed muscle obliterating the marrow cavity and areas of callus formation at both the outer and inner cortical surfaces. Histologically, graft incorporation was occurring at the outer cortical surface of the control allografts. In contrast, graft incorporation was occurring at both the outer and inner cortical surfaces of the allografts with an intramedullary muscle flap. The articular surface of the control allografts was severely degenerated. In allografts with an intramedullary muscle flap, the articular surface was smoother. Joints reconstructed with allografts with an intramedullary muscle flap had a significantly better range of motion at 5 weeks compared with control allografts. These results suggest that an intramedullary muscle flap can improve the functional results of joints reconstructed with partially demineralized and lyophilized osteochondral allografts by providing both vascularity and an increased population of mesenchymal stem cells capable of responding to bone morphogenetic proteins that reside in the partially demineralized allograft.

Animals↗
PubMed1997

Free flap coverage of the hand.

Microvascular free tissue transfer has been a major advancement in the treatment of soft-tissue defects of the hand. Free tissue transfers have expanded our options and have altered our approach to hand defects. It is no longer satisfactory to cover hand wounds with unsightly, bulky flaps of tissue. Microsurgical free tissue transfers have given us the tools for more refinement in hand soft-tissue reconstruction and have changed the standards for a successful outcome.

Hand↗
PubMed1996

A thioredoxin reductase-class of disulphide reductase in the protozoan parasite Giardia duodenalis.

We describe the purification and characterisation of a thioredoxin reductase-like disulphide reductase from the ancient protozoan parasite, Giardia duodenalis. This dimeric flavoprotein contains 1 mol FAD per subunit and had an apparent subunit molecular mass of 35 kDa. The purified enzyme catalysed the NADPH-dependent (Km = 8 microM) reduction of 5,5'-dithio-bis(2-nitrobenzoic acid) to thionitrobenzoate and was unable to utilise NADH as an electron donor. The sulphydryl-active compounds, N-ethylmaleimide, sodium arsenite and Zn2+ ions, strongly inhibited the enzyme suggesting that a thiol component forms part of the active site. Purified enzyme was able to utilise a variety of substrates, including cystine and oxidised glutathione, which suggests that it is a broad-range disulphide reductase, probably accounting for the majority of thiol cycling activity in this organism. While the G. duodenalis enzyme does not require an intermediate electron transport protein, analogous to thioredoxin, for activity, we have identified a candidate carrier protein which enhances DTNB turnover six fold, therefore implying that Giardia contains a thioredoxin-like system. Physical, enzymatic and spectral properties of the G. duodenalis disulphide reductase are also consistent with it being a member of the thioredoxin reductase-class of disulphide reductases. Furthermore, the internal amino acid sequence of a tryptic peptide generated from the purified protein was highly homologous with thioredoxin reductases from other sources. This is the first report of a disulphide reductase to be purified from the anaerobic protozoa and explains the so called "glutathione-induced thiol-reductase activity' previously observed in G. duodenalis.

Animals↗
PubMed1996

A H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis.

We describe the purification of a H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. The enzyme is a monomeric flavoprotein containing flavin adenine dinucleotide in a 1:1 molar ratio with the polypeptide. The NADH oxidase has an apparent molecular mass of 46 kDa and was homogenous as determined by denaturing gel electrophoresis and N-terminal amino acid sequencing. NADPH could substitute for NADH as an electron donor with a K(m) value of 4.2 microM for NADH and 16 microM for NADPH (pH 7.8 at room temperature). With oxygen as the primary electron acceptor under aerobic conditions, the pure enzyme did not produce O.-2 nor H2O2 as stoichiometric products of oxygen reduction, implicating H2O as the end product and obviating the need for superoxide dismutase. The ability to utilise oxygen explains the apparent respiration of the amitochondrial fermentative metabolism of Giardia. Mercurials, flavoantagonists and heavy metals (Cu2+ and Zn2+) inhibited this activity. Under anaerobic conditions the enzyme catalysed electron transfer at lower efficiencies to other electron acceptors including nitroblue tetrazolium, potassium ferricyanide, FAD and FMN, using either NADH or NADPH as electron donors. NADPH, however, was a more efficient electron donor. Cytochrome c was not reduced under any assay conditions used. The enzyme reduced the nitrofuran drugs, furazolidone (an antigiardial) and nitrofurantoin, to their toxic radical forms as determined by EPR. Metronidazole, a nitroimidazole, was not reduced. Pure NADH oxidase did not demonstrate ferredoxin:NAD(P)1 oxidoreductase activity since it could not accept electrons from reduced ferredoxin to regenerate NAD(P)H. The G. duodenalis NADH oxidase may, therefore, function as a terminal oxidase, similar to the mitochondrial cytochrome oxidase, and in the maintenance of an optimum intracellular redox ratio. This report of a flavoenzyme from Giardia places Giardia close to the anaerobic bacteria in evolutionary terms.

Amino Acid Sequence↗
PubMed1996

Bcl-2 expression inhibits prostaglandin E2-mediated apoptosis in B cell lymphomas.

Apoptosis is a critical mechanism in the maturation and maintenance of the immune system. However, the process by which cells die remains poorly understood. The proto-oncogene bcl-2 is considered important in determining whether cells enter an apoptotic pathway or survive. In this report, we first examined the differential sensitivity of immature (CH31) and mature (CH12) B cell lymphomas to growth inhibition by PGE2. The CH31 cell line was growth inhibited and underwent apoptosis in response to PGE2, unlike its mature counterpart, CH12. Furthermore, endogenous levels of the anti-apoptotic protein Bcl-2 in CH31 cells were low compared with CH12. To further investigate the role of Bcl-2 in PGE2- and cAMP-mediated cell death, a retroviral vector bearing the human bcl-2 gene was introduced into CH31. High expression of Bcl-2 in CH31 had no effect on growth inhibition induced by PGE2 or dibutyryl cAMP. In contrast, increased expression of Bcl-2 completely inhibited PGE2- and cAMP-mediated DNA fragmentation and nuclear condensation. Finally, cell cycle analysis of Bcl-2-expressing CH31 cells demonstrated that PGE2 increased the percentage of cells in G1, and analysis of synchronized populations revealed that PGE2 acts at all phases of the cell cycle to delay normal progression. These results support the hypothesis that apoptosis induced through PGE2 and cAMP signaling is sensitive to regulation by Bcl-2 in CH31 B cell lymphomas. Furthermore, unlike apoptosis, regulation of PGE2- and cAMP-mediated growth inhibition in B lineage cells is a distinct and Bcl-2-independent mechanism.

Apoptosis↗
PubMed1996

An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues.

We describe a new method for random mutagenesis of DNA based on the use of a mixture of triphosphates of nucleoside analogues. The method relies on DNA amplification in vitro with Taq polymerase and in the presence of the 5'-triphosphates of 6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C] [1,2]oxazin-7-one(dP) and of 8-oxo-2' deoxyguanosine (8-oxodG). The newly synthesised triphosphate derivative of dP (dPTP) is an excellent substrate for Taq polymerase (Km = 22 microM versus Km = 9.5 microM for TTP); it is incorporated in place of TTP and, with a approximately fourfold lower efficiency, in place of dCTP. After 30 cycles of DNA amplification, equimolar mixtures of the four normal dNTPs and dPTP yield the following frequencies of the four transition mutations: A-->G (4.4 x 10(-2), T-->C (4.3 x 10(-2), G-->A (1.1 x 10(-2) and C-->T (1.0 x 10(-2). The triphosphate derivative of 8-oxodG (8-oxodGTP) is incorporated opposite template adenine and yields two transition mutations (A-->C and T-->G) at frequencies of 0.8 x 10(-2) and 1.2 x 10(-2) respectively. Reaction mixtures containing dPTP and 8-oxodGTP results in both dP and 8-oxodG-induced mutations and an extensive array of codon changes in the absence of insertions and deletions. The method described differs from previous mutagenesis procedures in three respects: (1) it enables very high frequencies of base substitutions (up to 1.9 x 10(-1) (2) it allows control of the mutational load via the number of DNA amplification cycles and (3) it yields both transition and transversion mutations. The procedure may find application in the generation of libraries of DNA and protein mutants from which species with improved or novel activities may be selected.

Amino Acid Sequence↗
PubMed1996

Local irrigation with tissue factor pathway inhibitor inhibits intimal hyperplasia induced by arterial interventions.

OBJECTIVE: To evaluate the capacity of local irrigation with tissue factor pathway inhibitor (TFPI) to inhibit vessels from neointimal lesion formation following intimectomy or balloon angioplasty. DESIGN: The common carotid arteries in New Zealand white rabbits were subjected to either intimectomy or balloon angioplasty. INTERVENTION: Before restoring blood flow, the lumina of the vessels were irrigated with 1 mL of Dulbecco phosphate-buffered saline either with TFPI (100 micrograms/mL [TFPI group, n = 10]) or without TFPI (control group, n = 10). MAIN OUTCOME MEASURES: The area of neointimal formation and the ratio of the intimal to medial areas (I/M) were determined from elastin-stained sections. RESULTS: The area of neointima and the I/M ratio were not significantly different at 2 weeks postoperatively. However, at 4 weeks, TFPI-treated vessels demonstrated a significant reduction in the neointimal lesion and the I/M ratio compared with those of controls, following both angioplasty and intimectomy. Transmission electron microscopy showed a lack of platelet aggregation and thrombus formation at the intimal surface in the TFPI-treated vessels. CONCLUSIONS: Local irrigation with TFPI at the time of arterial interventional therapy inhibits intimal hyperplasia following either balloon angioplasty or intimectomy. We hypothesize that TFPI binds to the injured vessel surface and inhibits the cascade of thrombotic events that promote intimal hyperplasia.

Angioplasty, Balloon↗
PubMed1996

A molecular analysis of PGE receptor (EP) expression on normal and transformed B lymphocytes: coexpression of EP1, EP2, EP3beta and EP4.

The E-series prostaglandins (PGEs) are complex lipid regulators of B lymphocyte function. They inhibit the growth of certain B lymphoma lines. We report that heterogeneity with respect to PGE-induced growth inhibition correlates with the maturation state of the B cell lines. Specifically, the pre-B cell line 70Z/3 and the immature lymphoma CH31 are extremely sensitive to PGE2. To a lesser degree, other immature lymphomas (CH33, ECH408.1 and WEHI-231) are sensitive to PGE2. More mature lymphomas (BAL-17, CH12 and CH27) and fully differentiated myelomas (J558 and MOPC-315) are insensitive to PGE2. It is unknown what subtype of PGE receptor(s) (EPs) are expressed by B lymphocytes. It is also unknown if modulation of EP receptor expression could account for the differences in the sensitivity of these B cell lines to PGE2. To investigate these issues, reverse transcriptase polymerase chain reaction, Northern blot and DNA sequencing analyses were employed to obtain a definitive EP receptor subtype profile for these B cell lines, and for normal splenic B lymphocytes. Both normal and transformed B lymphocytes express mRNA encoding EP1, EP3beta and EP4 subtypes of PGE receptors. The B lineage cells do not express EP3alpha nor EP3gamma mRNA. The B cell lines are clonal, indicating that EP1, EP3beta and EP4 mRNA are coexpressed. Surprisingly, quantitative differences in basal EP1, EP3beta and EP4 expression were not observed between B cell lines despite their differing susceptibilities to PGE-induced growth inhibition. Conversely, the polyclonal activator LPS selectively upregulates EP4 mRNA expression in the mature B cell line CH12, but not in the LPS-sensitive pre-B cell line, 70Z/3. The activator LPS does not affect EP1 nor EP3beta mRNA expression. Treatment with dbcAMP, an analog of cAMP, mimics PGE-induced growth inhibition indicating that Gs-coupled EP2 and/or EP4 receptors mediate this inhibitory signal. Indeed, EP2 agonists mimic PGE2-induced growth inhibition unlike IP, EP1 and EP3-selective agonists. These data indicate that EP2 receptors are sufficient for mediating PGE-induced growth inhibition of susceptible B lineage cells.

Alprostadil↗
PubMed1996

Intratumoral radioimmunotherapy of a human colon cancer xenograft using a sustained-release gel.

Low tumor uptake and normal tissue toxicity limit the efficacy of RIT for the treatment of solid tumors. In this study, an intratumoral injectable gel drug delivery system for local administration of RIT was evaluated using the LS174T human colon cancer xenograft model in SCID mice. The injectable gel is a collagen-based drug delivery system designed for intratumoral (i.t.) administration, which has previously been shown to enhance drug retention at the injection site and reduce systemic drug exposure. We compared the local (tumor) retention and biodistribution of 111In-labeled NR-LU-10 monoclonal antibody given i.t. in the injectable gel versus simple aqueous solution. 111In gel given i.t. and 111In-NR-LU-10 given intraperitoneally (i.p.) were used as controls. The results showed that tumors treated with 111In-NR-LU-10 gel maintained the highest levels of radioactivity for up to 96 h. At 48 h after the administration of 111In-NR-LU-10 gel i.t., 111In-NR-LU-10 solution i.t., 111In gel i.t., or 111In-NR-LU-10 i.p., the level of radioactivity remaining in each gram of tumor was 98, 49, 45, and 16% of the injected dose, respectively. It was estimated that if 100 microCi of 90Y-NR-LU-10 were administered similarly, tumor treated with 90Y-NR-LU-10 gel i.t. would receive a dose of 90.0 Gy, whereas normal tissues in the same animal would receive a dose of approximately 2.43 Gy. In contrast, if 90Y-NR-LU-10 were delivered i.p., a comparable tumor would receive a dose of 16.8 Gy and corresponding normal tissues would receive 3.36 Gy. Consistent with these estimates, enhanced antitumor efficacy was observed when 90Y-NR-LU-10 gel was administered i.t. Tumor growth delay time was 6.9-fold (P < 0.01) longer in these animals (14.4 days) than in animals treated with 90Y-NR-LU-10 i.p. (2.1 days). Systemic toxicity was also significantly reduced in gel-treated animals as monitored by loss of body weight. This study demonstrated that intratumoral delivery of 90Y-NR-LU-10 gel markedly increased the retention of the radioisotope in tumors, enhanced the antitumor efficacy, and reduced systemic toxicity compared to systemic administration of the radiolabeled antibody. This injectable gel drug delivery system may allow for improvement in the therapeutic index for RIT.

Animals↗
PubMed1996

You can be in Jeopardy.

Clinical nurse educators are challenged continually to develop interesting and meaningful education sessions for staff members. At the Mayo Medical Center, Rochester, Minn, clinical nurse educators in the surgical services department created an education session based on the television game show Jeopardy. Staff members found they could remember information presented during the education session more easily because of the game show format.

Humans↗
PubMed1996

Treatment of avascular ulcers with cytokine-induced tissue generation and skin grafting.

BACKGROUND: Recombinant platelet-derived growth factor (rPDGF-BB) stimulates migration and proliferation of fibroblasts and smooth muscle cells and induces the rapid development of granulation tissue. This study investigated the use of rPDGF-BB and skin grafting to heal avascular ulcers using a rabbit model. We further investigated the effect of a hyaluronic acid carrier and a vascular pedicle on rPDGF-induced tissue generation in this model. METHODS: Large avascular ulcers were created on both ears of New Zealand white rabbits. One ulcer was treated with topical rPDGF-BB, the other with control buffer. After 5 or 7 days, the amount of granulation tissue migration from the wound margin was measured, and the wounds were skin grafted. In another group of ulcers, rPDGF-BB treatment was combined with a hyaluronic acid disk or an intact central axial ear artery and vein. RESULTS: Whereas control wounds generated 1.00 +/- 0.27 mm and 1.75 +/- 0.25 mm of granulated tissue migration from the wound margin by days 5 and 7, respectively, rPDGF-BB treatment increased the amount of granulation tissue migration to 1.88 +/- 0.23 mm and 3.00 +/- 0.86 mm by days 5 and 7 after treatment, respectively (P <0.05 at both time points). Hyaluronic acid disks stimulated 2.50 +/- 0.22 mm of granulation tissue migration by day 7 in controls; when rPDGF-BB was added to the carrier, the migration increased to 4.50 +/- 0.29 mm from the wound margin (P<0.05). Granulation tissue migration was further increased with an intact vascular pedicle: 5.75 +/- 0.48 mm in controls versus 7.75 +/- 0.25 mm with rPDGF-BB treatment (P<0.01). Skin grafts applied to the treated ulcers failed to survive in all groups except those with intact vascular pedicles. CONCLUSIONS. This study demonstrates that rPDGF-BB can increase the amount of granulation tissue generated over an avascular wound. This tissue generation is enhanced by both a hyaluronic acid carrier and a vascular pedicle. A vascular pedicle was required for skin graft survival. This study supports the role of hyaluronic acid in rPDGF-BB induced tissue generation and the requirement of a direct blood supply for functional cytokine-induced tissue generation.

Animals↗
PubMed1996

A new prenylated flavonol from the root of Petalostemon purpureus.

A new prenylated flavonol, petalopurpurenol (1), and a known dihydroflavonol, petalostemumol (2), have been isolated by DNA scission-guided fractionation of the organic portion of a 20% MeOH/CHCl3/H2O partition of a 50% MeOH/CHCl3 extract of the roots of Petalostemon purpureus. Compound 2 displayed moderate activity in DNA-scission assay. Both compounds 1 and 2 were evaluated for cytotoxicity in a panel of human cancer cell lines. The structures of petalopurpurenol (1) and petalostemumol (2) were determined by spectroscopic analysis.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Renal disease susceptibility and hypertension are under independent genetic control in the fawn-hooded rat.

Hypertension, diabetes and hyperlipidemia are risk factors for life-threatening complications such as end-stage renal disease, coronary artery disease and stroke. Why some patients develop complications is unclear, but only susceptibility genes may be involved. To test this notion, we studied crosses involving the fawn-hooded rat, an animal model of hypertension that develops chronic renal failure. Here, we report the localization of two genes, Rf-1 and Rf-2, responsible for about half of the genetic variation in key indices of renal impairment. In addition, we localize a gene, Bpfh-1, responsible for about 26% of the genetic variation in blood pressure. Rf-1 strongly affects the risk of renal impairment, but has no significant effect on blood pressure. Our results show that susceptibility to a complication of hypertension is under at least partially independent genetic control from susceptibility to hypertension itself.

Animals↗
PubMed1996

Drug retention following intravesical delivery of fluorouracil therapeutic adhesive in C3H mouse bladder.

We have developed a fibrinogen-based, sustained-retention drug delivery system, therapeutic adhesive (TA), for application to resected tumor beds to reduce local tumor recurrences. In this study we evaluated the feasibility, safety and retention of the TA formulated with 5-fluorouracil (5-FU TA) after intravesical administration in a mouse bladder model. Radiolabeled [14C]5-FU TA or [14C]5-FU solution was delivered intravesically to C3H/He female mice. After drug administration, retention of 14C in the bladder was quantified by storage-phosphor autoradiography. A 2.6-fold increase in retention was observed with 5-FU TA when compared with 5-FU solution. The AUC(2 min-5 h) for 5-FU TA was 685 nmol h/mm2 compared with 260 nmol h/mm3 for 5-FU solution. No signs of toxicity in the bladder tissue or treatment-associated adverse effects were observed in the mice.

Administration, Intravesical↗