Biomedical subjects

M Kelley

Publications and source records attributed to M Kelley.

At least 19 recordsLinked to original sources

PubMed1995

Cytotoxicity of BMS-181174. Effects of hypoxia, dicoumarol, and repair deficits.

The mitomycin C (MC) analog BMS-181174 (previously designated as BMY25067) has been shown to be active against a variety of solid tumors in mice. The activity of this compound against tumor cell lines resistant to MC and the different toxicity profiles of BMS-181174 and MC suggested that there may be significant differences in the metabolism and the mechanisms of action of these two compounds. Our studies with a mouse mammary tumor cell line (EMT6), a wild-type Chinese hamster cell line (AA8), and three repair-deficient Chinese hamster cell lines (UV4, UV5, and EM9) supported this concept. BMS-181174 was more toxic to all five cell lines in air than in hypoxia; in contrast, MC is more toxic in hypoxia. Dicoumarol (which increases the cytotoxicity of MC in hypoxia and reduces the cytotoxicity of this drug in air) did not alter the cytotoxicity of BMS-181174. This finding suggests that neither DT-diaphorase nor cytochrome b5 reductase is involved in the activation of BMS-181174. Studies with the repair-deficient cell lines suggest that DNA strand breaks are not important to the cytotoxicity of BMS-181174, and that cross-links and adducts may be the critical lesions; these studies also suggest that the lethal lesions produced by BMS-181174 are the same under aerobic and hypoxic conditions.

Animals↗

PubMed1995

Damnacanthal is a highly potent, selective inhibitor of p56lck tyrosine kinase activity.

Damnacanthal, an anthraquinone isolated from a plant extract, was found to be a potent, selective inhibitor of p56lck tyrosine kinase activity. The structure, potency, and selectivity of damnacanthal were confirmed by independent synthesis and testing. Damnacanthal exhibited an IC50 of 17 nM for inhibition of p56lck autophosphorylation and an IC50 of 620 nM for phosphorylation of an exogenous peptide by p56lck. Damnacanthal had > 100-fold selectivity for p56lck over the serine/threonine kinases, protein kinase A and protein kinase C, and > 40-fold selectivity for p56lck over four receptor tyrosine kinases. It also demonstrated modest (7-20-fold), but highly statistically significant, selectivity for p56lck over the homologous enzymes p60src and p59fyn. Mechanistic studies demonstrated that damnacanthal was competitive with the peptide binding site, but mixed noncompetitive with the ATP site. Although damnacanthal contains a potentially reactive aldehyde moiety, equilibrium dialysis experiments demonstrated that significant amine formation between damnacanthal and amines occurred only at high concentrations of reactants. However, damnacanthal appeared to bind nonspecifically to membrane lipids and was not active in whole cell tyrosine kinase assays. Damnacanthal is the most potent, selective inhibitor of p56lck tyrosine kinase activity described to date and may represent the starting point for the identification of novel, selective inhibitors of p56lck which are active in whole cell as well as in cell-free systems.

Alkaloids↗

PubMed1995

Endotoxin receptors (CD14) are found with CD16 (Fc gamma RIII) in an intracellular compartment of neutrophils that contains alkaline phosphatase.

CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein on the surfaces of monocytes and polymorphonuclear leukocytes (PMN) that binds and initiates cellular responses to bacterial LPS. PMN also contain an intracellular pool of CD14 that can be deployed rapidly to the cell surface in response to stimulation with a variety of agonists. To determine which of the well-characterized subcellular compartments of PMN contains CD14, cells were cavitated and fractionated on Percoll gradients. The gradient fractions were assayed for CD14 by ELISA and Western blot and for the marker proteins beta-glucuronidase (azurophil granules), vitamin B12 binding protein (specific granules), alkaline phosphatase (secretory vesicles and plasma membrane), and HLA (plasma membrane). Approximately one-half of the CD14 ran with plasma membrane fractions and one-half with intracellular membranes of light density. Both intracellular and cell surface CD14 were associated tightly with membrane, and both forms showed identical electrophoretic mobility. The intracellular CD14 was clearly not present in azurophil granules or specific granules, but ran precisely with alkaline phosphatase, a marker for secretory vesicles. Parallel studies showed that an additional GPI-linked protein, Fc gamma RIII (CD16), also fractionated precisely with CD14 and alkaline phosphatase. Association of CD14 with secretory vesicles were confirmed by studies on cells stimulated with the formyl peptide fNLLP for 20 min at 37 degrees C before fractionation. This treatment caused translocation of CD14 from intracellular fractions to plasma membrane fractions. No release of the specific granule marker vitamin B12 binding protein was observed under these conditions, whereas two other GPI-anchored proteins, alkaline phosphatase and CD16, moved coincidentally with CD14 to comigrate with the plasma membrane. Time course studies of CD14 and CD16 surface expression confirmed the rapid and coordinate up-regulation of these proteins. Thus, the intracellular compartment containing CD14 and CD16 had the properties of secretory vesicles. These vesicles may represent a specialized membrane domain of PMN enriched in GPI-anchored proteins.

Alkaline Phosphatase↗

PubMed1995

The use of combination internal fixation and hybrid external fixation in severe proximal tibia fractures.

Forty-eight patients with 50 severe fractures of the proximal tibia were followed prospectively for 2-4 years (mean 2.7 years) to evaluate the use of limited internal fixation combined with external fixation in the treatment of these injuries. There were 27 men and 21 women ranging in age from 20 to 74 years. Fractures were classified according to the A0 system (Mast J, Ganz R, Jacob R: Planning and reduction technique in fracture surgery. Berlin, Springer-Verlag, 1989), which included 5 A3, 6 C1, 16 C2, and 23 C3 fractures. All patients in this series healed; 48 fractures healed in an average of 12 weeks without subsequent surgery. There were two (4%) nonunions requiring bone graft. The average hospital special surgery knee score was 90 (68-100). Grading criteria for anatomical outcome revealed there were 17 (34%) excellent results, 24 (48%) good results, 6 (12%) fair results, and 3 (6%) poor results. In conclusion, this treatment method is associated with a high percentage of good and excellent results. Combined internal and external fixation combines the advantages of anatomic, stable fixation with less soft-tissue dissection and eliminates the need for large implants.

Adult↗

PubMed1995

Elevated circulating interleukin-7 levels in patients with systemic juvenile rheumatoid arthritis.

OBJECTIVE: Since interleukin-7 (IL-7) has been shown to induce monocyte production of IL-1, IL-6, IL-8, and tumor necrosis factor-alpha, we investigated plasma and synovial fluid levels of IL-7 in patients with juvenile rheumatoid arthritis (JRA). METHODS: IL-7 levels were measured using an ELISA in 60 patients with JRA and in 25 patients with other rheumatic disorders (ORD). RESULTS: In patients with systemic JRA, but not in patients with polyarticular or pauciarticular JRA or with ORD, plasma IL-7 levels were significantly higher (p < 0.0001) than those of healthy controls. IL-7 was undetected in all synovial fluid tested. Among patients with systemic JRA, those with persistent systemic symptoms had IL-7 levels significantly higher (p < 0.01) than those of patients in which systemic symptoms subsided. CONCLUSION: Plasma IL-7 levels are elevated in patients with systemic JRA and are associated with the presence and severity of systemic symptoms.

Adolescent↗

PubMed1994

Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14.

CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.

Acute-Phase Proteins↗

PubMed1992

Structural comparison between the mitochondrial aralkyl-CoA and arylacetyl-CoA N-acyltransferases.

The aralkyl and arylacetyl transferases were purified to homogeneity from bovine kidney by a slight modification of a previous procedure. The M(r) of the arylacetyl transferase was estimated to be 33,500 by SDS/PAGE and that of the aralkyl transferase to be 33,750 by a combination of SDS/PAGE and gel-filtration analysis. N-Terminal-sequence analysis indicated a blocked N-terminus for the arylacetyl transferase and gave the following sequence for the aralkyl transferase: M-F-L-L-Q-G-A-Q-M-L-Q-M-L-E-K. Amino acid analysis revealed differences in composition between the two enzymes. Most notable was the fact that the aralkyl transferase had more methionine and leucine. This difference could be partially accounted for by assuming that the methionine-and-leucine-rich N-terminus was missing from the arylacetyl transferase. Chemical cleavage of the two enzymes at methionine residues using CNBr gave rise to several peptides for each enzyme. N-Terminal-sequence analysis of the 8000-M(r) peptide from the arylacetyl transferase gave a sequence with 69% similarity to the 9000-M(r) peptide from the aralkyl transferase. This was taken to indicate a common origin for the two enzymes.

Acetyltransferases↗

PubMed1992

Administration of human recombinant IL-7 to normal and irradiated mice increases the numbers of lymphocytes and some immature cells of the myeloid lineage.

In vitro experiments performed by several investigators have demonstrated that IL-7 is a growth factor for immature B lymphocytes, thymocytes, and mature T lymphocytes. To evaluate the potential therapeutic use for human rIL-7 (rhuIL-7) as a hematopoietin, we have studied the in vivo hematopoietic effects of rhuIL-7 in mice. In these experiments, sublethally irradiated and normal mice were treated with or without rhuIL-7 for up to 26 days. Administration of rhuIL-7 significantly increased the white blood cell count in the peripheral blood and spleen in both normal and irradiated mice. Treatment with rhuIL-7 also accelerated lymphocytic recovery in irradiated mice. Precursor and mature B lymphocytes showed the greatest expansion in response to rhuIL-7 administration, with smaller increases in T lymphocytes being observed. In mice recovering from high dose irradiation, rhuIL-7 treatment resulted in preferential expansion of CD8+ T lymphocytes and more rapid normalization of the CD4/CD8 ratios. Differential analysis of peripheral blood smears demonstrated that rhuIL-7 also increased the numbers of immature granulocytes in both normal and irradiated mice. Moreover, administration of rhuIL-7 to normal, irradiated, cyclophosphamide-pretreated, or 5-fluorouracil-pretreated mice increased the number of acetylcholinesterase-positive megakaryocytes in the spleen, but not the bone marrow. Therefore, although the major in vivo effects of rhuIL-7 were on cells of the lymphocytic lineage, rhuIL-7 also increased the numbers of some immature cells of the myeloid lineage.

Animals↗

PubMed1992

Development of a shielded 241Am applicator for continuous low dose rate irradiation of rat rectum.

This paper describes the development of sources, applicators, and techniques that can be used to irradiate rat rectums with continuous irradiation at dose rates of interest in brachytherapy, either with the full circumference of the rectum irradiated, or with half of the circumference shielded from the radiation. The system uses encapsulated 241Am sources, to irradiate rat rectum with 60 keV photons continuously at a dose rate of up to 50 cGy/hr. Details of the design and fabrication of the 241Am sources, the rectal applicator, the dosimetry of the system, and the protocols for preparing and irradiating the rats, and for detecting early rectal injury using histological examination of irradiated rectum are presented. Highly effective shielding (attenuation factors as low as 0.04) of half of the circumference of the rat rectum was achievable. Unidirectional 241Am irradiators for intracavitary brachytherapy offer a unique tool for examining the effects of shielding a portion of the circumference of the rat rectum, on the radiation tolerance of the rectum.

Americium↗

PubMed1992

Effects of hyperbaric oxygen and a perfluorooctylbromide emulsion on the radiation responses of tumors and normal tissues in rodents.

Perfluorochemical emulsions are being examined in many laboratory and clinical studies as possible adjuncts to radiotherapy and chemotherapy. The studies reported here examine the clinical potential of hyperbaric oxygen (HBO) in combination with a highly concentrated perfluorochemical emulsion (Oxygent) containing 100% w/v perfluorooctylbromide (PFOB). HBO alone produced only a small improvement in the radiation response of BA1112 tumors in WAG/rij rats, while regimens combining HBO with Oxygent produced much greater radiation sensitization. A sham emulsion, formulated without the O2-carrying PFOB, did not alter the radiation response of the tumors in comparison with that seen with HBO alone. Neither HBO nor Oxygent plus HBO altered the radiosensitivity of bone marrow progenitor cells in BALB/c mice. HBO alone augmented skin reactions in BALB/c mice, but addition of Oxygent did not alter the skin reactions in comparison to those seen with HBO alone. Regimens combining Oxygent with HBO selectively increased the radiation sensitivity of tumors relative to normal tissues, thereby enhancing the therapeutic ratio. These results support the potential usefulness of perfluorochemical emulsions and HBO in clinical radiation therapy.

Animals↗

PubMed1992

Effects of the perfluorochemical emulsion FMIQ on the radiation response of EMT6 tumours.

The effects of FMIQ, a perfluorochemical emulsion based on perfluoro-N-methyldecahydroisoquinoline, were examined using BALB/c mice and EMT6 mammary carcinomas. The radiobiological effects of FMIQ were similar to those found previously for Fluosol in the same tumour/host system. Although the perfluorochemical content (20% w/v) and oxygen-carrying capacity of FMIQ are similar to those of Fluosol, the formulation of FMIQ offers some advantages over that of Fluosol. For example, FMIQ has greater stability during storage. FMIQ also is formulated without pluronic F-68 and is based on a perfluorochemical (FMIQ) having a shorter tissue dwell time than the perfluorotripropylamine in Fluosol; it therefore may produce fewer side-effects than Fluosol. The lifetime of the circulating perfluorochemical droplets in BALB/c mice was longer than FMIQ than for Fluosol; this could offer an advantage in fractionated radiotherapy. These findings give reason to expect that FMIQ may prove to be a better emulsion than Fluosol for clinical use as an adjunct to cancer therapy.

Animals↗

PubMed1992

Preclinical evaluation of Oxygent as an adjunct to radiotherapy.

These studies examine the potential value of a concentrated emulsion of perfluorooctylbromide (perflubron; Oxygent, Alliance Pharmaceutical Corp.) as an adjunct to radiotherapy. The effects of Oxygent on solid tumors were examined using EMT6 mammary tumors in BALB/c mice and BA1112 rhabdomyosarcomas in WAG/rij rats. Treatment with Oxygent plus O2, carbogen (95% O2/5% CO2), or hyperbaric oxygen (HBO) increased the effects of radiation on the tumors. Analyses of tumor cell survival curves and measurements of intratumor pO2 showed that this potentiation reflected an increase in the proportion of well-oxygenated tumor cells. Neither treatment of the animals with carbogen, O2, or HBO alone nor treatment of air-breathing rodents with Oxygent produced changes of similar magnitude. Treatment with a vehicle emulsion containing all the components of Oxygent except the perflubron did not alter tumor radiosensitivity, showing that tumor radiosensitization required the oxygen-transporting perfluorocarbon, and did not result from any biologic or physiologic effects of other components of the emulsion. These studies also examined the effects of Oxygent on the radiation responses of mouse skin and bone marrow. Oxygent selectively increased the radiation sensitivity of tumors relative to these normal tissues, thereby increasing the therapeutic ratio and producing therapeutic gain. Oxygent appears to warrant further testing as an adjunct to cancer therapy.

Animals↗

PubMed1991

A coupled assay for bile acid:CoA ligase.

A new assay for the enzyme bile acid:CoA ligase is presented. The new assay is designed to supplant the existing radiometric assays which require radiolabeled bile acids. The new assay couples the formation of bile acid-CoA to its glycination in a reaction catalyzed by bile acid-CoA:glycine N-acyltransferase. The coupling reaction utilizes [14C]glycine and the bile acid-CoA is quantitatively converted to [14C]glycobile acid. The [14C]glycobile acid is isolated by solvent extraction and quantitated by liquid scintillation counting. The method is shown to be accurate, highly sensitive, and applicable to a wide variety of bile acids.

Acyltransferases↗

PubMed1991

Modulation of tumor oxygenation and radiosensitivity by a perfluorooctylbromide emulsion.

The effect of a concentrated perfluorooctylbromide emulsion (Oxygent) on the radiosensitivity and oxygenation of solid tumors was examined using EMT6 mammary tumors in BALB/c mice and BA1112 rhabdomyosarcomas in WAG/rij rats. Treatment with Oxygent plus carbogen or oxygen breathing increased the radiosensitivity of both tumors. Analysis of tumor cell survival data and polarographic measurements of intratumoral pO2 indicated that this potentiation reflected an increase in the proportion of well-oxygenated tumor cells. Treatments with carbogen breathing alone, with Oxygent plus air-breathing, or with a vehicle emulsion containing all the components except the perfluorocarbon did not produce comparable improvements in tumor radiosensitivity. Concentrated perfluorooctylbromide emulsions appear to warrant further development and preclinical testing as adjuncts to cancer therapy.

Animals↗

PubMed1991

In vitro methodology for resistance arteries.

Two commonly used methods for examining the physiological and pharmacological properties of isolated resistance arteries are the ring-mounted preparation and the cannulated, pressurized vessel. Each technique is discussed and consideration given to limitations and advantages. Also presented are examples of comparative differences between them, and practical experimental schemes for calibrating cannulae and for perfusing resistance arteries. Although both methods are valuable, the cannulated approach may better reflect the in vivo properties of the arteries.

Animals↗

PubMed1991

Activity of C-7 substituted cyclic acetal derivatives of mitomycin C and porfiromycin against hypoxic and oxygenated EMT6 carcinoma cells in vitro and in vivo.

A series of cyclic acetal derivatives of mitomycin C (MC) and porfiromycin (POR) were tested for their ability to kill hypoxic and oxygenated EMT6 tumor cells. Amino methyl acetal and thioacetal substitutions at C-7 of MC and POR dramatically increased the cytotoxicity of the compounds to hypoxic EMT6 tumor cells in vitro but had little effect on the aerobic toxicities. In contrast, a methyl substitution at N1a markedly decreased the aerobic cytotoxicities of the compounds but did not alter the hypoxic cytotoxicities. The POR acetal, BMY-42355, had the largest differential between hypoxic and aerobic cytotoxicities yet observed among MC analogs. Preliminary studies in mice showed that BMY-42355 had good antineoplastic activity when used alone or in combination with radiation and was less toxic than POR; the therapeutic ratio of this compound in these initial studies was higher than those of either MC or POR.

Animals↗

PubMed1990

A test of the tridimensional personality theory: association with diagnosis and platelet imipramine binding in obsessive-compulsive disorder.

We administered the Tridimensional Personality Questionnaire (TPQ) to a sample of 25 individuals with obsessive-compulsive disorder (OCD) and 35 normal controls. As predicted, OCD cases scored much higher on the harm avoidance dimension than normal controls. Findings for the novelty seeking and reward dependence dimensions were less dramatic, although compatible with the underlying theory. Despite a theoretical link between the harm avoidance dimension and serotonin-mediated neuropathways, we failed to find an association between this dimension and platelet imipramine binding in either OCD cases or controls.

Arousal↗

PubMed1990

Effects of cytochrome P-450 monooxygenase inducers on mouse hepatic microsomal metabolism of testosterone and alkoxyresorufins.

The effects of treatment with phenobarbital, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), pregnenolone-16 alpha-carbonitrile (PCN), 3-methylcholanthrene (3-MC) and isosafrole on the hepatic microsomal formation of nine monohydroxy metabolites of testosterone and the O-dealkylation of the ethyl and pentyl ethers of resourfin were evaluated in adult male C57BL/6J and DBA/2NCR mice. In both strains, phenobarbital, TCPOBOP and PCN induced testosterone 2 beta-, 6 beta-, 15 beta- and 16 beta-hydroxylases up to 5-fold, while phenobarbital and TCPOBOP increased the rate of dealkylation of pentoxyresorufin by approximately 30-fold. However, phenobarbital and TCPOBOP did not exhibit identical patterns of induction for the testosterone oxidation reactions. Hepatic microsomes from C57BL/6J mice treated with TCPOBOP displayed a depression in 6 alpha-testosterone hydroxylase activity, which was also observed in PCN-treated animals, whereas phenobarbital-treated mice exhibited an elevation in this monooxygenase activity. A dose of TCPOBOP (0.5 mumol/kg) previously demonstrated to represent an ED50 for mouse aminopyrine N-demethylase activity was also found to approximate the ED50 for pentoxyresorufin O-dealkylase activity in the C57BL/6J mouse. Isosafrole or 3-MC treatment had little effect on testosterone metabolism or pentoxyresorufin O-dealkylase activity in either strain, while 3-MC induced ethoxyresorufin O-deethylase activity in C57BL/6J but not DBA/2NCR mice. This study confirms that TCPOBOP is a potent cytochrome P-450 inducer which most closely resembles phenobarbital in its mode of action. However, TCPOBOP and phenobarbital do not evoke identical modulations of cytochrome P-450-dependent monooxygenases in mice.

Animals↗