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Biomedical subjects

D E Kuhn

Publications and source records attributed to D E Kuhn.

18 recordsLinked to original sources
PubMed2001

Iron transport into mycobacterium avium-containing phagosomes from an Nramp1(Gly169)-transfected RAW264.7 macrophage cell line.

Nramp1 is an important determinant of innate resistance of macrophages to the growth of intracellular microorganisms. We previously showed that Nramp1 functions to transport iron from the cytoplasm into phagosomes of Mycobacterium avium-infected macrophages. The purpose of this investigation was to further characterize the factors that regulate Nramp1-mediated iron transport into phagosomes. Treatment of Nramp1(Gly169) macrophages with the lysomotrophic agents chloroquine or ammonium chloride reduced the import of iron significantly. We found that macrophage-activating cytokines, including TNF-alpha, IFN-gamma, IL-1alpha, and GM-CSF, when added prior to M. avium, increased the transport of iron into the phagosome. This increase in iron transport was not a result of an increased amount of Nramp1 protein in the phagosome nor to new protein synthesis. Treatment of Nramp1(Gly169)-transfected macrophages with inhibitors of protein kinase C (PKC) diminished the import of iron into the phagosomes. Iron import was inhibited by an anti-Nramp1 antibody against the putative fourth outer-loop region of Nramp1 but not by an anti-Nramp1 antibody against the carboxy terminus. The significance of these results on the orientation of Nramp1 in the phagosome membrane and on the transport of iron is discussed.

Animals↗
PubMed1999

Differential iron transport into phagosomes isolated from the RAW264.7 macrophage cell lines transfected with Nramp1Gly169 or Nramp1Asp169.

The transport of iron by RAW264.7 macrophage cell lines transfected with either Nramp1Gly169 (resistant) or Nramp1ASp169 (susceptible) alleles was assessed. We found no difference between resistant and susceptible cells in the rate of Fe import or export when Fe transport was measured in intact cells. In contrast, the rate of Fe import by latex-bead phagosomes isolated from resistant cells was more than double the rate by latex-bead phagosomes from susceptible cells. Similarly, phagosomes isolated from resistant cells that had been pre-labeled with 55Fe-citrate before phagocytosis contained up to four times as much Fe as the corresponding phagosomes from susceptible cells. Phagocytosis of Mycobacterium avium was accompanied by an increase in the production of hydroxyl radicals by Nramp1cGly169-transfected macrophages but not by macrophages transfected with the susceptible allele. These results are consistent with the hypothesis that Nramp1 functions to transport Fe into the bacterium-containing phagosome where it serves as a catalyst for the Haber-Weiss reaction, which accounts for the increased capacity of these cells to limit mycobacterial growth.

Animals↗
PubMed1999

Role of iron in Nramp1-mediated inhibition of mycobacterial growth.

Innate resistance to mycobacterial growth is mediated by a gene, Nramp1. We have previously reported that Nramp1 mRNA from macrophages of Mycobacterium bovis BCG-resistant (Bcgr) mice is more stable than Nramp1 mRNA from macrophages of BCG-susceptible (Bcgs) mice. Based on these observations and on reports that show that the closely related Nramp2 gene is a metal ion transporter, we evaluated the effect of iron on the growth of Mycobacterium avium within macrophages as well as on the stability of Nramp1 mRNA. The addition of iron to macrophages from Bcgs mice resulted in a stimulation of mycobacterial growth. In contrast, iron increased the capacity of macrophages from Bcgr mice to control the growth of M. avium. When we treated recombinant gamma interferon (IFN-gamma)-activated macrophages with iron, we found that iron abrogated the growth inhibitory effect of IFN-gamma-activated macrophages from Bcgs mice but that it did not affect the capacity of macrophages from Bcgr mice to control microbial growth. A more detailed examination of the effect of iron on microbial growth showed that the addition of small quantities of iron to resident macrophages from Bcgr mice stimulated antimicrobial activity within a very narrow dose range. The effect of iron on the growth inhibitory activity of macrophages from Bcgr mice was abrogated by the addition of catalase or mannitol to the culture medium. These results are consistent with an Fe(II)-mediated stimulation of the Fenton/Haber-Weiss reaction and hydroxyl radical-mediated inhibition of mycobacterial growth.

Animals↗
PubMed1999

Clarifying an ambiguous functional analysis with matched and mismatched extinction procedures.

Results of functional analysis were ambiguous in suggesting that self-injurious behavior (SIB) was maintained by escape, sensory reinforcement, or both. To help clarify these results, we compared escape extinction, sensory extinction, and the combined treatments. Sensory extinction proved to be a necessary and sufficient treatment, whereas escape extinction failed to decrease SIB. These analyses helped to clarify the function of SIB and to identify an effective and efficient treatment.

Adult↗
PubMed1998

The evaluation and treatment of aggression maintained by attention and automatic reinforcement.

In the current investigation, we used direct and indirect methods to assess and treat several topographies of aggression that were hypothesized to have separate operant functions in a young boy with severe mental ratardation and pervasive developmental disorder. First, a functional analysis of aggression, using the methods described by Iwata, Dorsey, Slifer, Bauman, and Richman (1982/1994), was conducted and produced inconclusive results. Next, indirect methods were used to develop a second functional analysis, which showed that chin grinding (firmly pressing and grinding his chin against the skin and bones of others) persisted independent of social contingencies and that the other topographies of aggression (e.g., hitting, kicking) were maintained by social positive reinforcement (attention). A treatment designed to decrease aggression maintained by attention--functional communication training with extinction--reduced all forms of aggression except chin grinding. This latter topography of aggression, which we hypothesized was maintained by automatic reinforcement, was reduced when the response--reinforcer relation was interrupted through response blocking and the child was provided with an alternative form of chin stimulation.

Aggression↗
PubMed1998

Establishing discriminative control of responding using functional and alternative reinforcers during functional communication training.

Functional communication training (FCT) is a popular treatment for problem behaviors, but its effectiveness may be compromised when the client emits the target communication response and reinforcement is either delayed or denied. In the current investigation, we trained 2 individuals to emit different communication responses to request (a) the reinforcer for destructive behavior in a given situation (e.g., contingent attention in the attention condition of a functional analysis) and (b) an alternative reinforcer (e.g., toys in the attention condition of a functional analysis). Next, we taught the participants to request each reinforcer in the presence of a different discriminative stimulus (SD). Then, we evaluated the effects of differential reinforcement of communication (DRC) using the functional and alternative reinforcers and correlated SDs, with and without extinction of destructive behavior. During all applications, DRC (in combination with SDs that signaled available reinforcers) rapidly reduced destructive behavior to low levels regardless of whether the functional reinforcer or an alternative reinforcer was available or whether reinforcement for destructive behavior was discontinued (i.e., extinction).

Adolescent↗
PubMed1996

Site-directed mutagenesis of monocyte chemoattractant protein-1 identifies two regions of the polypeptide essential for biological activity.

Monocyte chemoattractant protein-1 (MCP-1) mediates monocyte migration into tissues in inflammatory diseases and atherosclerosis. We have investigated structure-activity relationships for human MCP-1. Mutations were introduced based upon differences between MCP-1 and the structurally related but functionally distinct molecule interleukin-8 (IL-8). Mutant proteins produced using the baculovirus/insect cell expression system were purified and their ability to stimulate monocyte chemotaxis and elevation of intracellular calcium in THP-1 monocytic leukaemia cells was measured. Two regions in MCP-1 were identified as important for its biological activity. One region consists of the sequence Thr-Cys-Cys-Tyr (amino acids 10-13). Point mutations of Thr-10 to Arg and Tyr-13 to Ile greatly lowered MCP-1 activity. The second functionally important region is formed by Ser-34 and Lys-35. Insertion of a Pro between these two residues, or their substitution by the sequence Gly-Pro-His, caused nearly complete loss of MCP-1 activity. Competition binding experiments showed that the mutations that affected activity also lowered the ability to compete with wild-type MCP-1 for receptors on THP-1 cells. Point mutations at positions 8, 15, 30, 37, 38 and 68 had little effect on MCP-1 activity. The important regions that we have identified in MCP-1 correspond with previously identified functionally important regions of IL-8, suggesting that the two molecules bind to their respective receptors by similar contacts.

Amino Acid Sequence↗
PubMed1995

The cytomegalovirus US28 protein binds multiple CC chemokines with high affinity.

Human cytomegalovirus encodes several proteins with high similarity to seven transmembrane domain receptors. We investigated the ability of one of these proteins, the product of the US28 open reading frame, to bind various chemoattractant ligands. When transfected into COS-7 cells, the US28 product conferred high affinity binding to the labeled chemokines monocyte chemoattractant protein-1 (MCP-1) (Kd = 6.0 x 10(-10) M) and RANTES (Kd = 2.7 x 10(-10) M). Binding of these labeled ligands could be competed by the unlabeled macrophage inflammatory proteins MIP-1 alpha and MIP-1 beta, with Kd values in the range 1.2 x 10(-9) to 7.5 x 10(-9) M. Comparisons of the sequences of US28 and other receptors that bind chemokines should help to define regions responsible for receptor-ligand interactions.

Amino Acid Sequence↗
PubMed1993

Phosphoenolpyruvate and creatine phosphate augment ATP and magnesium-dependent, Fc epsilon RI-mediated activation of phospholipase C in RBL cell ghosts.

We have previously reported that Fc epsilon RI-mediated activation of PLC occurs in plasma membrane vesicles derived from RBL cells. This activity is dependent on ATP and magnesium, and is greatly enhanced by the addition of either PEP or CP. We undertook these studies to determine whether these compounds augment Fc epsilon RI-mediated activation of PLC because of their ability to form ATP. The specific findings were 1) the addition of increasing amounts of ATP to the ghost vesicles cannot substitute for the combination of ATP and either PEP or CP; 2) the addition of increasing amounts of ATP or a combination of ATP and either PEP or CP results in similar levels of intravesicular ATP; 3) many metabolically related compounds have some activity to support receptor-mediated PI hydrolysis, but only PEP, CP, and 3-PG give a maximal signal; and 4) the inability of ATP alone to support optimal receptor-mediated PI hydrolysis does not appear to be due to the accumulation of ADP is only modestly inhibitory. Taken together, these data are evidence that augmentation of Fc epsilon RI-mediated PI hydrolysis by PEP and CP is not explained simply by the ability of these compounds to generate intravesicular ATP.

Adenosine Triphosphate↗
PubMed1993

Fc epsilon RI-mediated hydrolysis of phosphoinositides in permeable membrane vesicles.

We have used hypotonic lysis of cytoplasts derived from rat basophilic leukemia (RBL) cells to prepare organelle and cytoplasm-depleted membrane vesicles called 'RBL cell ghosts' (Dreskin and Metzger, 1991). Unlike other membrane preparations, the RBL ghosts hydrolyze phosphoinositides (PIs) in response to aggregation of the high affinity receptor for IgE (Fc epsilon RI) and have proven useful for studies of the molecular events involved in transduction of this signal. A significant limitation of these preparations is that they are sealed. Thus, to incorporate membrane-impermeant molecules (such as ATP) into the intravesicular space of the ghosts, they must be added as the ghosts are formed. We have now overcome this problem by permeabilizing the ghosts with alpha-toxin from S. aureus and find that, following permeabilization, ghosts activated via Fc epsilon RI, hydrolyze PIs for a longer time than do non-permeabilized ghosts. As in the intact ghosts, this response is absolutely dependent upon ATP and is enhanced by the addition of either phosphoenolpyruvate (PEP) or creatine phosphate (CP). This report demonstrates that we can now manipulate the intravesicular environment of the RBL ghosts and extends the utility of these preparations as a model system for the study of signal transduction following activation via Fc epsilon RI.

Adenosine Triphosphate↗
PubMed1990

Cholesterol metabolism in dystrophic mice. II. Altered enzyme activities.

In a previous study we found that free cholesterol (FC) and cholesterol ester (CE) concentrations in fast-glycolytic (FG) muscle tissue from dystrophic mice are significantly higher than normal. This increase is not due to an increased capacity for de novo cholesterol biosynthesis. HMG-CoA reductase (HMGR) (the enzyme which catalyzes the rate limiting step) activity is significantly decreased in dystrophic muscle compared to normal. This decrease is paralleled by an increased capacity for both CE production and hydrolysis, i.e., both Acyl-CoA:cholesterol acyltransferase (ACAT) activity and the activities of both lysosomal and sarcoplasmic cholesterol ester hydrolases (CEH) are greatly increased. These enzyme changes in dystrophic FG muscle are similar those observed in normal tissues with elevated levels of cholesterol, which suggests that such changes are not the cause of the altered cholesterol concentrations but are rather the response of the tissue to elevated levels of cholesterol.

Animals↗
PubMed1987

Fiber-specific cholesterol changes in murine dystrophy.

The cholesterol concentration in dystrophic mouse muscle is reported to be increased compared to normal. The muscles studied are, however, composed in most cases of more than one fiber type. As a result, the observed concentration increase may be due to a general increase or may be due to changes in the proportion of individual fiber types which themselves differ in cholesterol concentration. To decide between these possibilities we have measured the cholesterol concentrations (both free cholesterol and cholesterol esters) in normal and dystrophic whole gastrocnemius muscles and compared the values with the concentrations in fast-glycolytic muscle tissue alone. The cholesterol concentrations in both whole and fast-glycolytic sections of dystrophic muscle are increased compared to normal, with the largest increase in the cholesterol ester fraction. Furthermore, the concentration changes in fast-glycolytic fibers are due mainly to cholesterol ester differences in both membrane and sarcoplasm fractions, with differences in the latter being larger. The data show that changes in whole muscle concentrations cannot be ascribed solely to altered fiber type proportions.

Animals↗
PubMed1983

Aspects of hepatic cholesterol metabolism in normal and dystrophic chicken embryos.

Several aspects of hepatic cholesterol metabolism have been studied in normal and dystrophic chicken embryos (12 days in ovo). Dystrophic embryo livers weigh the same and have the same total protein content as their normal counterparts. However, the total cholesterol content of dystrophic embryo livers is significantly decreased compared with the content of normal embryo livers. This decrease in total cholesterol is due mainly to a large decrease in the amount of cholesterol esters, although the free cholesterol content is also decreased. The proportions of free cholesterol and cholesterol esters are 43 and 57%, respectively, in normal embryo livers, but this proportion is essentially reversed in dystrophic embryo livers (i.e., 56 and 44%). The decreased total cholesterol content of dystrophic embryo livers is not apparently due to a decrease in the rate of free cholesterol de novo biosynthesis or to a decrease in the rate of free cholesterol esterification. However, the decrease in cholesterol ester content may be due to an increase in the rate of its hydrolysis, as a result of increased levels of sterol ester hydrolase in dystrophic livers. These data are consistent with the proposal that membranes in dystrophic tissues are altered as a result of altered cholesterol metabolism and utilization.

Animals↗
PubMed1981

Altered acetylcholinesterase isozyme patterns in mice with hereditary muscular dystrophy.

Normal and dystrophic mouse muscles were separated into a predominantly white muscle fraction (gastrocnemius, extensor digitorum longus) and a predominantly red muscle fraction (diaphragm). Acetylcholinesterase (AChE) was extracted from each muscle fraction using a Triton X-100/NaCl buffer. Six forms of AChE were separated from each muscle homogenate by velocity sedimentation on linear sucrose gradients. Their apparent sedimentation coefficients in each case were 19.7S, 16.0S, 13.3S, 10.4S, 7.6S, and 3.9S. Gel electrophoresis of crude muscle homogenates under nondenaturing conditions (native gels) and of ech separate isozyme fraction gave one band of AChE activity with a consistent Rf (relative mobility) value. Reelectrophoresis of native gel bands on SDS/acrylamide slab gels revealed a similar monomeric subunit protein from either crude muscle homogenates or isozyme fractions with an apparent molecular weight of approximately 69,000 daltons. Our results indicate that the AChE distribution and activity are severely affected in dystrophic "white" muscles (anaerobic) but much less so in "red" muscles (aerobic). Dystrophic predominantly white muscles weigh less, contain less protein, and have a decreased total AChE activity in comparison with their normal counterparts. Furthermore, the relative proportions of AChE activity in each isozyme fraction is altered between normal white and dystrophic white muscle fractions: i.e., dystrophic white muscle contains a decreased proportion of a low molecular weight form (7.6S) and increased proportions of higher molecular weight forms (16.0S, 19.7S). In contrast, no significant differences occur in AChE activity or distribution between normal and dystrophic predominantly red muscle. The changes in white muscle AChE are toward a pattern common to red muscle. This suggests that the effect of muscular dystrophy and its related stress on mouse white muscle is at least in part a shift from a predominantly anaerobic, fatigable metabolism to an aerobic, fatigue-resistant metabolism.

Acetylcholinesterase↗
PubMedDate not supplied

Noncontingent reinforcement: effects of satiation versus choice responding.

Recent research findings suggest that the initial reductive effects of noncontingent reinforcement (NCR) schedules on destructive behavior result from the establishing effects of an antecedent stimulus (i.e., the availability of "free" reinforcement) rather than extinction. A number of authors have suggested that these antecedent effects result primarily from reinforcer satiation, but an alternative hypothesis is that the individual attempts to access contingent reinforcement primarily when noncontingent reinforcement is unavailable, but chooses not to access contingent reinforcement when noncontingent reinforcement is available. If the satiation hypothesis is more accurate, then the reductive effects of NCR should increase over the course of a session, especially for denser schedules of NCR, and should occur during both NCR delivery and the NCR inter-reinforcement interval (NCR IRI). If the choice hypothesis is more accurate, then the reductive effects of NCR should be relatively constant over the course of a session for both denser and leaner schedules of NCR and should occur almost exclusively during the NCR interval (rather than the NCR IRI). To evaluate these hypotheses, we examined within-session trends of destructive behavior with denser and leaner schedules of NCR (without extinction), and also measured responding in the NCR interval separate from responding in the NCR IRI. Reductions in destructive behavior were mostly due to the participants choosing not to access contingent reinforcement when NCR was being delivered and only minimally due to reinforcer satiation.

Adolescent↗
PubMedDate not supplied

Partially reassembled high density lipoproteins. Effects on cholesterol flux, synthesis, and esterification in normal human skin fibroblasts.

The effects of a partially reassembled high density lipoprotein (PR-HDL) prepared by a detergent removal method on the cholesterol metabolism of fibroblasts has been tested under conditions of lipid depletion. The PR-HDL were composed of apolipoprotein A-I (1 mol) from human plasma HDL, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) 100 mol, and varying amounts of cholesterol. Below 15 mol%, the PR-HDL effected a net efflux of cholesterol from the fibroblasts; as a consequence, the activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A acyltransferase, respectively, were increased and decreased. Above 15 mol% cholesterol in the PR-HDL, net influx of cholesterol was observed and 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity was decreased, while acyl coenzyme A acyltransferase activity was increased. The uptake of several phosphatidylcholines, apo A-I, and cholesterol were measured as a function of time. The rates of lipid transfer to the cells decreased with increasing hydrophobicity of the transferring lipid. By contrast, in parallel experiments a relatively small quantity of 125I apo A-I was associated with the cells. Our results suggest that a component of lipid transfer from PR-HDL to fibroblasts occurs via monomeric transfer through the aqueous phase that is independent of apo A-I binding.

Cells, Cultured↗