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Biomedical subjects

M K Brown

Publications and source records attributed to M K Brown.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of transforming growth factor beta signaling in MCF-7 cells results in resistance to tumor necrosis factor alpha: a role for Bcl-2.

Transforming growth factor beta (TGF-beta) is a multifunctional cytokine capable of regulating diverse cellular processes. In this study we investigated the effect of autocrine TGF-beta signaling on tumor necrosis factor (TNF) alpha-induced cell death. We abrogated the TGF-beta autocrine loop by overexpression of a truncated TGF-beta type II receptor in MCF-7 breast carcinoma cells and found that this generated resistance to TNF-alpha-induced cytotoxicity. To elucidate the molecular basis of the influence of TGF-beta on TNF-alpha-induced cytotoxicity, we evaluated the expression levels or activities of proteins involved in TNF-alpha signal transduction or the regulation of apoptosis in general in TGF-beta-responsive and TGF-beta-nonresponsive MCF-7 cells. We observed no significant difference in the expression of TNF-alpha receptors or the TNF receptor-associated death domain protein. In addition, downstream activation of nuclear factor kappaB by TNF-alpha was not altered in cells that had lost TGF-beta responsiveness. Analysis of members of the Bcl-2 family of apoptosis-regulatory proteins revealed that Bcl-X(L) and Bax expression levels were not changed by disruption of TGF-beta signaling. In contrast, the TGF-beta-nonresponsive cells expressed much higher levels of Bcl-2 protein and mRNA than did cells with an intact TGF-beta autocrine loop. Furthermore, restoration of a TGF-beta signal to MCF-7 cells that had spontaneously acquired resistance to TGF-beta caused a reduction in Bcl-2 protein expression. Taken together, our data indicate that loss of autocrine TGF-beta signaling results in enhanced resistance to TNF-alpha-mediated cell death and that this is likely to be mediated by derepression of Bcl-2 expression.

Apoptosis↗
PubMed2000

gamma-glutamyltranspeptidase-deficient knockout mice as a model to study the relationship between glutathione status, mitochondrial function, and cellular function.

gamma-Glutamyltranspeptidase (GGT)-deficient mice (GGT(-/-)) display chronic glutathione (GSH) deficiency, growth retardation, and die at a young age (<20 weeks). Using livers from these mice, we investigated the relationship between GSH content, especially mitochondrial, and mitochondrial and cellular function. We found that the GSH content of isolated liver mitochondria was diminished by >/=50% in GGT(-/-) mice when compared with wild-type mice. Respiratory control ratios (RCRs) of GGT(-/-) mice liver mitochondria were </=60% those of wild-type mice primarily as a result of impaired state 3 respiration. Mitochondrial adenine nucleotide content was decreased by >/=40% in mitochondria obtained from GGT(-/-) mice. We observed a strong correlation between mitochondrial GSH content and RCRs. Even moderate decreases (<50%) correlated with adverse effects with respect to respiration. Electron microscopy revealed that livers from GGT(-/-) knockout mice were deprived of fat and glycogen, and swollen mitochondria were observed in animals that were severely deprived of GSH. Thus, GGT(-/-) mice exhibit a loss of GSH homeostasis and impaired oxidative phosphorylation, which may be related to the rate of adenosine triphosphate (ATP) formation and subsequently leads to progressive liver injury, which characterizes the diseased state. We also found that supplementation of GGT(-/-) mice with N-acetylcysteine (NAC) partially restored liver GSH, but fully restored mitochondrial GSH and respiratory function. Electron microscopy revealed that the livers of NAC-supplemented GGT(-/-) mice contained fat and glycogen; however, slightly enlarged mitochondria were found in some livers. NAC supplementation did not have any beneficial effect on the parameters examined in wild-type mice.

Adenosine Diphosphate↗
PubMed2000

Mechanical and histologic evaluation of Collagraft in an ovine lumbar fusion model.

The purpose of this study was to determine the effectiveness of a composite material composed of Type I bovine dermal collagen, 65% hydroxyapatite, and 35% tricalcium phosphate ceramic (Collagraft Bone Graft Matrix Strip NeuColl Incorporated, Palo Alto, CA) as a bone graft substitute for spinal fusion with and without the use of autologous bone marrow in an ovine lumbar spine model with pedicle screw fixation. Twenty-four adult sheep underwent a single level posterolateral (intertransverse process) L3-L4 lumbar fusion with one of three graft materials combined with rigid pedicle screw fixation. The three graft materials were Collagraft, Collagraft with marrow, and autogenous corticocancellous bone graft. Animals were euthanized 6 months after surgery and evaluated using dual energy x-ray absorptiometry, radiographs, histologic analysis, and mechanical testing. Dual energy xray absorptiometry between the transverse processes revealed that the mineral densities for the two Collagraft groups were significantly higher than the autogenous bone graft group. Histologic analysis confirmed that Collagraft was highly compatible and was well incorporated into the fusion mass. Both Collagraft groups had thick trabeculae and a mixture of lamellar and plexiform bone. The autogenous bone graft group had a smaller fusion complex, composed primarily of lamellar bone with thinner and fewer trabeculae. All three groups had similar mechanical properties. These results support the use of Collagraft in spinal fusion with pedicle screw fixation.

Absorptiometry, Photon↗
PubMed1999

Comparison of the efficacy of 7-hydroxystaurosporine (UCN-01) and other staurosporine analogs to abrogate cisplatin-induced cell cycle arrest in human breast cancer cell lines.

DNA-damaging agents such as cisplatin arrest cell cycle progression at either the G1, S, or G2 phase, although the G1 arrest is seen only in cells expressing the wild-type p53 tumor suppressor protein. Caffeine has been shown to abrogate the S and G2 arrest in p53-defective cells and to enhance cytotoxicity, but at concentrations too toxic to administer to humans. We have reported that 7-hydroxystaurosporine (UCN-01) also overcomes S and G2 phase arrest and enhances the cytotoxicity of cisplatin. We show here that UCN-01 at non-cytotoxic concentrations abrogated S and G2 arrest induced by cisplatin in two p53-defective human breast cancer cell lines. UCN-01 pushed the cells through S phase and mitosis, with subsequent apoptosis. Inhibition of mitosis with nocodazole reduced the apoptosis induced by UCN-01 plus cisplatin. Seven staurosporine analogs were compared for their ability to abrogate cell cycle arrest. Staurosporine was as effective as UCN-01 at abrogating S and G2 arrest, but the concentrations required were cytotoxic. K252a abrogated S phase arrest but failed to abrogate G2 arrest because alone it induced G2 arrest. Hence, K252a did not enhance cisplatin-induced cytotoxicity because it failed to push the cells through a lethal mitosis. None of the other analogs influenced cell cycle progression at the concentrations tested. Accordingly, UCN-01 was the only analog that overcame cell cycle arrest and enhanced the cytotoxicity of cisplatin while exhibiting no cytotoxicity of its own. Hence, UCN-01 remains the most promising candidate for testing clinically in combination with cisplatin.

Alkaloids↗
PubMed1999

Biocompatibility and degradation of collagen bone anchors in a rabbit model.

Bone anchors are used to fasten tendons and ligaments to bone during reconstructive surgery. Although metal anchors are often used, an anchor that could resorb and permit normal bone regeneration would be advantageous. The objective of the study was to evaluate the biocompatibility and degradation of bone anchors that consist of collagen-based bodies, ceramic washers, and polyester sutures. Eighteen rabbits underwent bilateral implantations in the distal femoral condyles. Nine animals received glutaraldehyde-crosslinked fibrillar collagen bone anchors (FC) and nine received glutaraldehyde-crosslinked fibrillar collagen bone anchors containing tricalcium phosphate (FC-TCP). Three animals per group were sacrificed at postimplantation weeks 1, 6, and 12. One femur from each rabbit was evaluated histologically, and the contralateral side underwent biomechanical pull-out testing. Histological evaluation of the implant site indicated that the FC and FC-TCP bone anchors were both biocompatible. The FC-TCP formulation degraded earlier than the FC formulation, and FC-TCP showed significant degradation at 6 weeks; the FC and FC-TCP formulations both showed similar amounts of degradation at 12 weeks. The degrading anchor bodies appeared to be osteoconductive as evidenced by new bone ingrowth into the degrading collagen matrices without a fibrous interface. These results suggest that collagen-based bone anchors have potential as bioresorbable orthopedic implants.

Animals↗
PubMed1999

Ultrasonic enhancement of antibiotic action on Escherichia coli biofilms: an in vivo model.

Biofilm infections are a common complication of prosthetic devices in humans. Previous in vitro research has determined that low-frequency ultrasound combined with aminoglycoside antibiotics is an effective method of killing biofilms. We report the development of an in vivo model to determine if ultrasound enhances antibiotic action. Two 24-h-old Escherichia coli (ATCC 10798) biofilms grown on polyethylene disks were implanted subcutaneously on the backs of New Zealand White female rabbits, one on each side of the spine. Low-frequency (28.48-kHz) and low-power-density (100- and 300-mW/cm2) continuous ultrasound treatment was applied for 24 h with and without systemic administration of gentamicin. The disks were then removed, and the number of viable bacteria on each disk was determined. At the low ultrasonic power used in this study, exposure to ultrasound only (no gentamicin) caused no significant difference in bacterial viability. In the presence of antibiotic, there was a significant reduction due to 300-mW/cm2 ultrasound (P = 0.0485) but no significant reduction due to 100-mW/cm2 ultrasound. Tissue damage to the skin was noted at the 300-mW/cm2 treatment level. Further development of this technique has promise in treatment of clinical implant infections.

Animals↗
PubMed1999

Access to health care: Louisiana minor consent statutes.

The Louisiana minor's consent statutes enable minors to consent for medical treatment, emergency treatment, treatment of sexually-transmitted diseases, and treatment of substance abuse. The legislative intent for permitting minors to consent to treatment without the express consent of a parent or guardian was to provide minors access to high-quality health care services in order to encourage the betterment of the health and welfare of the citizens of our state. A minor cannot consent for an abortion or sterilization. The statutes do not allow a minor to refuse treatment consented to by his parent or guardian. However, needed medical treatment can be provided to a consenting minor over the objections of a parent or guardian. Where a minor can consent, confidentiality from parent's or guardian's knowledge is permitted but not assured; confidentiality is granted at the discretion of the physician or medical staff.

Adolescent↗
PubMed1998

Biliary secretion of alpha-tocopherol and the role of the mdr2 P-glycoprotein in rats and mice.

The mechanism by which alpha-tocopherol (alpha-T) is secreted into the bile is not known; however, we have previously demonstrated that treatment with piperonyl butoxide (PIP, 1 g/kg) results in increased biliary output of both alpha-T and phosphatidylcholine within 3 h of ip injection in rats and that the biliary output of both substances was prevented by chemicals that disrupt microtubules (Toxicol. Appl. Pharmacol. 139, 411-417 (1996)). The P-glycoprotein (Pgp) encoded by the mdr2 gene has been shown to transport phosphatidylcholine into the bile; therefore, in the current study, we utilized the Pgp inhibitor verapamil to investigate the possible involvement of Pgps in the biliary secretion of alpha-T. When rats were iv injected with verapamil (4 mg/kg) 10 min prior to PIP treatment, verapamil prevented the PIP-induced increases in biliary alpha-T and phosphatidylcholine output and resulted in biliary alpha-T outputs that were significantly less than controls. Also, we determined that the biliary alpha-T levels in mdr2 knockout mice were 25% of those in wildtype mice; furthermore, mdr2 liver, lung, and kidney levels of alpha-T and glutathione differed from those of wildtype. To investigate the fate of biliary alpha-T, we injected 14C-labeled alpha-T into the bile duct cannulae of rats and determined that approximately 60% of the radioactivity was reabsorbed within 1 h. Our results indicate that alpha-T undergoes enterohepatic circulation and that the biliary secretion of alpha-T, basally and following chemical treatment, is dependent on the presence of a functioning mdr2 Pgp in rats and mice.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1997

Hypodermoclysis therapy. In a chronic care hospital setting.

Occasionally, elderly patients experience acute, episodic incidents of illness that result in dehydration or a high potential for dehydration (e.g., flu, diarrhea). At times, patients may be unable, or refuse, to take fluids orally. Enteral routes via a nasogastric tube or enteral stomach tube may also not be available. In the past, these patients often had to be transferred from home or long-term care facilities to an acute care hospital for intravenous therapy. A transfer of the acutely ill elderly patient to an acute care hospital is often very stressful to the patient and his/her family and is costly to the health care delivery system. Hypodermoclysis, the process of rehydrating a patient by providing isotonic fluids into the subcutaneous tissues over a short time period, provides an alternative method to deal with acute, short-term fluid deficit problems in the elderly. Hypodermoclysis therapy can be administered in a chronic care setting thus potentially decreasing the need to transfer the elderly client to an acute care hospital. The purpose of this study was to investigate the use of hypodermoclysis therapy in solving acute, or potentially acute fluid deficit problems, that were anticipated to be both reversible and short term in nature. This was carried out in an elderly population that resided in a 284-bed chronic care hospital in southern Ontario.

Acute Disease↗
PubMed1996

The involvement of protein phosphatases in the activation of ICE/CED-3 protease, intracellular acidification, DNA digestion, and apoptosis.

Many events in apoptosis have been identified but their temporal relationships remain obscure. Apoptosis in human ML-1 cells induced by etoposide is characterized by intracellular acidification, enhanced Hoechst 33342 fluorescence, DNA digestion, chromatin condensation, and proteolysis of poly(ADP-ribose) polymerase. This proteolysis is a marker for the action of ICE/CED-3 proteases, which are critical activators of apoptosis. We observed that three serine/threonine protein phosphatase inhibitors, okadaic acid, calyculin A, and cantharidin, prevented all of these apoptotic characteristics. To determine which protein phosphatase was involved, we investigated the dephosphorylation of the retinoblastoma susceptibility protein Rb, a substrate for protein phosphatase 1 but not protein phosphatase 2A. Rb was dephosphorylated during apoptosis, and each inhibitor prevented this dephosphorylation at the same concentrations that prevented apoptosis. No increase in protein phosphatase 1 activity was observed in apoptotic cells suggesting that dephosphorylation of Rb may result from loss of Rb kinase activity in the presence of a constant level of protein phosphatase activity. Long term inhibition of protein phosphatase 1 (>8 h) also led to the appearance of dephosphorylated Rb, cleavage of poly(ADP-ribose) polymerase and apoptosis, suggesting these events are not solely dependent upon protein phosphatase 1. Rb dephosphorylation was also observed in several other models of apoptosis. Hence, an imbalance between protein phosphatase 1 and Rb kinase may be a common means to activate ICE/CED-3 proteases resulting in the subsequent events of apoptosis.

Acids↗
PubMed1996

Colchicine and vinblastine prevent the piperonyl butoxide-induced increase in rat biliary output of alpha-tocopherol.

alpha-Tocopherol, a lipid-soluble chain-breaking antioxidant, is important in the protection of biologic membranes and is transported in the body in association with lipids. It has been estimated that the liver contains up to 29% of the alpha-tocopherol stores of the body. Piperonyl butoxide (PIP) is a widely used synthetic methylenedioxyphenyl insecticide synergist. Bile was collected for 1 hr prior to, and 5 hr following, i.p. injection of 1 g/kg PIP. Within 3 hr of PIP administration, biliary alpha-tocopherol output increased to 132% of pretreatment measurements. Hepatic alpha-tocopherol levels of PIP-treated rats decreased to 26% below those of controls at the end of the bile collection period. Biliary bile acid and phospholipid output also increased significantly following PIP treatment. In a second set of experiments, rats were pretreated with either colchicine or one of two doses of vinblastine; both colchicine and the higher dose of vinblastine prevented the PIP-induced decrease in hepatic alpha-tocopherol and the increase in biliary output of alpha-tocopherol, bile acids, and phospholipids. These results suggest that (i) PIP-induced biliary secretion of alpha-tocopherol is associated with altered biliary phospholipid output, (ii) a significant portion of hepatic alpha-tocopherol may function as a highly mobilizable source of antioxidant for possible rapid enterohepatic distribution, and (iii) microtubules may be involved in this process.

Animals↗
PubMed1991

Modification of hepatic vitamin E stores in vivo. II. Alterations in plasma and liver vitamin E content by 1,2-dibromoethane.

Previous studies with methyl ethyl ketone peroxide (MEKP), a radical generator, showed depletion of plasma vitamin E and liver glutathione (GSH) levels prior to a decrease of liver vitamin E levels. Since hepatic pools of this vitamin may serve to maintain circulating levels of vitamin E under conditions of oxidative challenge, we have evaluated the similarity of response after treatment with 1,2-dibromoethane (DBE), a compound that is not known to generate oxyradicals or to induce lipid peroxidation in vivo. Treatment of normal rats with DBE caused a depletion in hepatic vitamin E levels 1 day after treatment; however, in contrast to our prior findings with MEKP this depletion after DBE treatment was observed in tandem with elevations in the plasma content of vitamin E. Liver vitamin E depletion was neither dependent upon a sustained liver GSH depletion nor upon hepatocellular death. Mobilization and export of hepatic vitamin E did not result in an immediate whole body redistribution of this vitamin in that pulmonary and renal levels of vitamin E remained normal under conditions of liver vitamin E depletion. Moreover, the stimulus that resulted in exportation of liver vitamin E was maintained by daily treatments with DBE. DBE caused a substantial elevation above control values in liver GSH content and these elevations were also maintained by daily DBE treatments. In experiments to assess the influence of prandial replacement of vitamin E on the extent of depletion in response to DBE treatment, rats were fed a vitamin E-deficient diet for 2 days prior to treatment. This short pulse of a vitamin E-deficient diet delayed (to 2 days) both the elevation in liver GSH content and the depletion of liver vitamin E and hastened (to 1 day) the elevation in plasma vitamin E concentration. These observations suggest the presence of at least two pools of liver vitamin E and that one of these pools, which comprises at least 30% of the total hepatic vitamin E content, is able to be mobilized and exported in response to chemical challenge. The stimulus that resulted in liver vitamin E exportation in response to DBE treatment seems to result from wholly intrahepatic processes and may not be a direct response to lipid peroxidation. Moreover, the similarity between the time-course and the extent of hepatic vitamin E depletion observed after treatment with either MEKP or DBE suggests a similarity in physiochemical processes that function to mobilize hepatic vitamin E stores.

Alanine Transaminase↗
PubMed1991

Role strain.

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Career Choice↗
PubMed1988

Improved screening for colorectal cancer by immunological detection of occult blood.

A radial immunodiffusion technique for detecting faecal haemoglobin and the Hemoccult II kit used with and without rehydration of the faecal sample were compared in a screening programme for bowel cancer, in which 1328 subjects took part. A positive result was obtained in 170 (13%) subjects. Nineteen of the 153 subjects investigated were found to have colorectal carcinomas and 52 had polyps (40 with adenomas). Radial immunodiffusion and Hemoccult II with and without rehydration detected bleeding in, respectively, all 19, 15, and 11 subjects with colorectal carcinoma. Hemoccult II with and without rehydration detected only seven and six, respectively, of 11 Duke's stage A carcinomas, whereas all 11 were detected with the immunological test. Hemoccult II with and without rehydration and radial immunodiffusion detected bleeding from adenomas in, respectively, 22, 14, and 34 of the 40 subjects. False positive results occurred in 55 out of 1302 subjects by Hemoccult II with rehydration, in 28 out of 1304 by Hemoccult II without rehydration, and in 50 out of 1304 by the immunological technique; true positive results were defined as bleeding from carcinomas and adenomas. Immunological detection of occult blood in faecal samples seems to show more adenomas and carcinomas (particularly early lesions) than the Hemoccult II kit and has a rate of false positive results that is acceptably low.

Adenoma↗
PubMed1985

Mechanism of chemical-induced toxicity. I. Use of a rapid centrifugation technique for the separation of viable and nonviable hepatocytes.

A major obstacle in defining the mechanism of chemical-induced toxicity has been the inability to distinguish between events that cause cell death and those that result from cell death. This problem results from measuring biochemical parameters in tissues or cell pellets containing both viable and nonviable cells. In the present study, we described a method for the rapid separation of viable hepatocytes from nonviable cells and medium prior to biochemical analysis. Separation of viable hepatocytes was accomplished in a microcentrifuge tube by layering a sample of isolated hepatocyte suspension over a dibutyl phthalate oil layer and centrifuging for several seconds. As a result, greater than 90% of the hepatocytes centrifuged through dibutyl phthalate were viable while greater than 90% of the cells recovered above the oil layer were nonviable. The separation of viable hepatocytes by the dibutyl phthalate method was not affected by the presence of the hepatotoxins, adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or ethyl methanesulfonate (EMS), though the ratio of viable to nonviable cells in the suspension was drastically reduced. The metabolic and morphological integrity of hepatocytes centrifuged through dibutyl phthalate was altered after cell suspensions were treated with the ADR-BCNU or EMS. These chemically treated viable hepatocytes showed degenerative ultrastructural changes and a greater than 80% reduction in intracellular K+ and glutathione concentrations. Because centrifugation through dibutyl phthalate does not significantly alter the concentration of intracellular constituents nor the ultrastructure of control hepatocytes, the signs of reversible injury observed in hepatocytes centrifuged through oil resulted from the chemical treatment. These data indicate that the dibutyl phthalate separation technique offers the advantage of monitoring only viable hepatocytes for changes in membrane integrity or metabolic performance during a toxic chemical insult.

Animals↗