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Biomedical subjects

R L Smith

Publications and source records attributed to R L Smith.

At least 109 records · Page 6Linked to original sources
PubMed1999

A replication-incompetent adenovirus vector with the preterminal protein gene deleted efficiently transduces mouse ears.

Adenoviruses offer great potential as gene therapy agents but are limited by the strong inflammatory response that occurs in response to the recombinant virus. Since the degree of inflammation correlates in part with the potential of the viral vector for replication, we constructed a preterminal protein (pTP) deletion mutant adenovirus type 5 vector, Ad5dl308DeltapTPbeta-gal, that is replication incompetent due to deletion of the pTP gene and that has the E1 genes replaced by the Escherichia coli lacZ reporter gene under the control of the cytomegalovirus major immediate-early promoter. This virus was compared with a first-generation, replication-defective adenovirus vector, Ad5dl308beta-gal, that is isogenic except that it contains a wild-type pTP gene. To examine transduction efficiency and induction of inflammation, we developed a novel system involving intradermal injection of BALB/c mouse ears. Mouse ears can be accurately measured to determine the degree of edema as an indirect measurement of inflammation. Edema and inflammation were induced in a dose- and time-dependent manner by both viruses and correlated well. LacZ activity correlated inversely with edema and inflammation. The pTP-defective vector Ad5dl308DeltapTPbeta-gal transduced mouse ears much more efficiently and induced edema and inflammatory cell infiltration approximately 10-fold less efficiently than the first-generation vector Ad5dl308beta-gal. The diminished inflammatory response and increased efficiency of transduction observed with Ad5dl308DeltapTPbeta-gal indicate its promise as a gene therapy agent for other tissues. The results also demonstrate that the mouse ear model offers potential for the study of adenovirus-induced inflammation because of the ready access of the ears, the relative ease of continuous measurement, and the sensitivity to adenovirus transducing vectors.

Adenoviruses, Human↗
PubMed1999

Induction of macrophage C-C chemokine expression by titanium alloy and bone cement particles.

Particulate wear debris is associated with periprosthetic inflammation and loosening in total joint arthroplasty. We tested the effects of titanium alloy (Ti-alloy) and PMMA particles on monocyte/macrophage expression of the C-C chemokines, monocyte chemoattractant protein-1 (MCP-1), monocyte inflammatory protein-1 alpha (MIP-1alpha), and regulated upon activation normal T expressed and secreted protein (RANTES). Periprosthetic granulomatous tissue was analysed for expression of macrophage chemokines by immunohistochemistry. Chemokine expression in human monocytes/macrophages exposed to Ti-alloy and PMMA particles in vitro was determined by RT-PCR, ELISA and monocyte migration. We observed MCP-1 and MIP-1alpha expression in all tissue samples from failed arthroplasties. Ti-alloy and PMMA particles increased expression of MCP-1 and MIP-1alpha in macrophages in vitro in a dose- and time-dependent manner whereas RANTES was not detected. mRNA signal levels for MCP-1 and MIP-1alpha were also observed in cells after exposure to particles. Monocyte migration was stimulated by culture medium collected from macrophages exposed to Ti-alloy and PMMA particles. Antibodies to MCP-1 and MIP-1alpha inhibited chemotactic activity of the culture medium samples. Release of C-C chemokines by macrophages in response to wear particles may contribute to chronic inflammation at the bone-implant interface in total joint arthroplasty.

Antibodies, Monoclonal↗
PubMed1999

Signaling pathways for tumor necrosis factor-alpha and interleukin-6 expression in human macrophages exposed to titanium-alloy particulate debris in vitro.

BACKGROUND: Loosening of the implant after total joint arthroplasty remains a serious problem. The activation of macrophages by wear debris from implants, mediated by the release of cytokines that elicit bone resorption, may lead to loosening. The purpose of the present study was to elucidate the mechanisms of macrophage activation by titanium particles from the components of implants and to identify the signaling pathways involved in particle-mediated release of cytokines. METHODS: Macrophages were isolated from mononuclear leukocytes obtained from healthy human donors and were exposed to titanium-alloy particles that had been obtained from periprosthetic membranes collected at revision total joint arthroplasties and then enzymatically prepared. The experimental protocols included examination of the effects of the inhibition of phagocytosis and the binding of antibodies to macrophage complement receptors on particle-induced macrophage activation. The release of the proinflammatory cytokines TNF-alpha (tumor necrosis factor-alpha) and IL-6 (interleukin-6) was used to assess macrophage activation. The signaling pathways involved in the induction of cytokine release were analyzed by identification of phosphorylated proteins with use of the Western blot technique and by translocation of the transcription factors nuclear factor-kappa B (NF-kappaB) and nuclear factor-interleukin-6 (NF-IL-6) into the nuclear protein fraction with use of electrophoretic mobility shift assays. The role of serine/threonine and tyrosine kinase pathways in the activation of nuclear factors and the release of cytokines was examined with use of selective pharmacological agents. RESULTS: Exposure of macrophages to titanium-alloy particles in vitro for forty-eight hours resulted in a fortyfold increase in the release of TNF-alpha and a sevenfold increase in the release of IL-6 (p<0.01). Phagocytosis of particles occurred in approximately 73 percent of the macrophages within one hour of exposure. Pretreatment of the macrophages with cytochalasin B reduced phagocytosis by 95 percent but did not reduce the release of TNF-alpha or IL-6. Thus, phagocytosis of particles was not necessary for induction of the release of TNF-alpha or IL-6 in the cultured macrophages. Ligation of the macrophage CD11b/CD18 receptors by integrin-specific antibodies also increased the release of TNF-alpha and IL-6. Antibodies to CD11b/ CD18 receptors (macrophage Mac-1 receptors) reduced phagocytosis of particles by 50 percent (p<0.05). (The CD11b/CD18 macrophage receptor is the macrophage receptor for the complement component CR3bi. The CD11b/CD18 macrophage receptor can also bind to ICAM-1 and ICAM-2. CD is the abbreviation for cluster of differentiation, and ICAM is the abbreviation for intercellular adhesion molecule.) Inhibition of phagocytosis was not accompanied by a decrease in the release of TNF-alpha and IL-6. Blocking RNA synthesis with actinomycin D or preventing protein synthesis with cycloheximide abolished or decreased particle-induced release of TNF-alpha and IL-6 from the macrophages. Macrophage release of TNF-alpha and IL-6 in response to particles coincided with increased tyrosine phosphorylation and mitogen-activated protein kinase activation. Inhibition of tyrosine and serine/threonine kinase activity decreased the particle-induced release of cytokines. Exposure of macrophages to either titanium-alloy particles or to antibodies to the receptor proteins CD11b and CD18 for thirty minutes activated the transcription factors NF-kappaB and NF-IL-6. Inhibition of particle phagocytosis did not block activation of the transcription factors. However, inhibition of tyrosine and serine/threonine kinase activity decreased the activation of NF-kappaB and NF-IL-6. CONCLUSIONS: These data suggest that particle induced macrophage release of TNF-alpha and IL-6 does not require phagocytosis but is dependent on tyrosine and serine/threonine kinase activity culminating in activation of

Alloys↗
PubMed1999

Carbon dioxide transport by proteic and facilitated transport membranes.

Membrane separation of gases is governed by the permeability of each species across the membrane. The ratio of permeabilities yields the selectivity. Use of certain organic carriers in facilitated transport membranes and the CO2 converting enzyme carbonic anhydrase (CA) in proteic and facilitated transport membranes allows a dramatic increase in CO2 selectivity over other gases. CA has a low Km (9 mM), which we predicted would allow it to scavenge CO2 to very low partial pressures. Our goal was to determine if CA could remove CO2 from an environment at levels of 0.1% or less. Prior measurements of CO2 transport across thin supported liquid membranes showed that addition of CA enhanced CO2 flux by 3- to 100-fold. Proteic films use bifunctional reagents (e.g., glutaraldehyde) to cross-link the enzyme forming a gel. Bovine serum albumin (BSA) is often added for structural stability. Using such a preparation we examined the ability of proteic films to improve CO2 selectivity and to scavenge CO2 from a mixed gas stream. Proof-of-concept results, measured by mass spectrometry, showed a fivefold improvement in CO2 capture rate with maximal improvement at CO2 values of 1% partial pressure difference in the presence of 0 atm absolute difference. At 0.1% CO2 the membrane exhibited a 76% improvement over controls. At 0.3% CO2 the improvement is about threefold. CA proteic membranes exhibit selectivity for CO2 over oxygen and nitrogen in excess of three orders of magnitude. A CA-based proteic or facilitated transport membrane should readily achieve CO2 partial pressures of 0.05% under CELSS conditions. In addition to proteic membranes we are exploring direct immobilization of engineered CA to ultra-high-permeability teflon membranes. Site-directed mutagenesis was used to add functional groups while retaining full enzymatic activity. These results provide a basis for development of far more efficient CO2 capture proteic and facilitated transport membranes with increased selectivity to values closer to 100-fold at 1% CO2. The result will be CO2 selectivity at 0.1% on the order of 400-fold. These results exceed those obtained with other technologies.

Biological Transport↗
PubMed1998

The flavin-containing monooxygenase 2 gene (FMO2) of humans, but not of other primates, encodes a truncated, nonfunctional protein.

Flavin-containing monooxygenases (FMOs) are NADPH-dependent flavoenzymes that catalyze the oxidation of heteroatom centers in numerous drugs and xenobiotics. FMO2, or "pulmonary" FMO, one of five forms of the enzyme identified in mammals, is expressed predominantly in lung and differs from other FMOs in that it can catalyze the N-oxidation of certain primary alkylamines. We describe here the isolation and characterization of cDNAs for human FMO2. Analysis of the sequence of the cDNAs and of a section of the corresponding gene revealed that the major FMO2 allele of humans encodes a polypeptide that, compared with the orthologous protein of other mammals, lacks 64 amino acid residues from its C terminus. Heterologous expression of the cDNA revealed that the truncated polypeptide was catalytically inactive. The nonsense mutation that gave rise to the truncated polypeptide, a C --> T transition in codon 472, is not present in the FMO2 gene of closely related primates, including gorilla and chimpanzee, and must therefore have arisen in the human lineage after the divergence of the Homo and Pan clades. Possible mechanisms for the fixation of the mutation in the human population and the potential significance of the loss of functional FMO2 in humans are discussed.

Alleles↗
PubMed1998

The CorA Mg2+ transport protein of Salmonella typhimurium. Mutagenesis of conserved residues in the third membrane domain identifies a Mg2+ pore.

The CorA transport system is the major Mg2+ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg2+ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. In contrast, mutation of Tyr292, Met299, and Tyr307 greatly altered the transport properties of CorA. Y292F, Y292S, Y292C, or Y292I mutations essentially abolished transport, without effect on expression or membrane insertion. M299C and M299A mutants exhibited a decrease in cation affinity for Mg2+, Co2+, or Ni2+ of 10-50-fold without a significant change in uptake capacity. Mutations at Tyr307 had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A mutations for Mg2+ and Co2+ was decreased 3-10-fold, while affinity for Ni2+ was unchanged compared with the wild type CorA. In contrast, the affinity of the Y307S mutant for all three cations was decreased 2-5-fold. Projection of the third transmembrane segment as an alpha-helix suggests that Tyr292, Met299, and Tyr307 all reside on the same face of the alpha-helix. We interpret the transport data to suggest that a hydroxyl group is important at Tyr307, and that these three residues interact with Mg2+ during transport, forming part of the cation pore or channel within CorA.

Amino Acid Sequence↗
PubMed1998

Effects of serum protein opsonization on cytokine release by titanium-alloy particles.

This study tested whether macrophages respond differently to retrieved titanium-alloy particles than they do to machined titanium-alloy particles and assessed whether pretreatment of machined titanium-alloy particles with human serum would influence macrophage activation and cytokine release in vitro. Human monocyte/macrophages were isolated from normal healthy donors and exposed to increasing concentrations of machined and retrieved titanium-alloy particles. The profile of cytokine release was determined by commercially available ELISA kits. Machined titanium-alloy particles were opsonized with human serum and added to macrophage cultures. Serum protein binding was confirmed by SDS-PAGE analysis. The results showed that machined titanium-alloy particles and retrieved titanium-alloy particles stimulate a similar level of cytokine release when tested at comparable concentrations. Opsonization of the machined particles with human serum increased the macrophage release of cytokines in the first 12 h after exposure compared to nonopsonized particles. At 24 h, the opsonized particles stimulated significantly higher levels of cytokine release, but only at the greatest particle concentrations. This study demonstrates that machined titanium alloy induces a metabolic response in macrophages similar to that of titanium-alloy particles retrieved from failed total hip arthroplasty. In addition, these data show that serum protein binding to orthopedic biomaterial debris alters the macrophage reaction to the particles.

Alloys↗
PubMed1998

Expression of mRNA for the elav-like neural-specific RNA binding protein, HuD, during nervous system development.

The expression of mRNA for the neuronal antigen HuD (Elavl4) associated with paraneoplastic encephalomyelitis and sensory neuronopathy was evaluated in the developing and adult rat nervous system. Using RNase protection assay and non-radioactive in situ hybridization histochemistry HuD expression was shown to be expressed at high levels at the earliest time point observed (E15), but declined significantly during the first postnatal week to levels which were maintained into adulthood. In the adult, HuD expression became restricted primarily to large pyramidal-like neurons. Exceptions of note were many smaller neurons within a variety of thalamic nuclei. Expression of HuD was observed to be coincident with terminal differentiation of all neuronal structures evaluated regardless of the timing of their development, providing correlative evidence for a role in neuronal differentiation or the maintenance of neuronal phenotype. The marked restriction of HuD mRNA expression with maturity suggests that its functional role in adult neurons varies significantly throughout the CNS.

Animals↗
PubMed1998

Ontogeny of cation-Cl- cotransporter expression in rat neocortex.

Neuronal precursors and immature cortical neurons actively accumulate Cl- and as a consequence depolarize in response to GABAA receptor activation. With maturity, intracellular Cl- decreases resulting in a shift towards GABAA inhibition. These observations suggest that changes in expression of cation-Cl- cotransporters may have a significant role in the ontogeny of neuronal Cl- homeostasis. Using ribonuclease protection analysis and in situ hybridization we examined the developmental expression of all presently known members of the cation-Cl- cotransporter gene family in rat brain. Of the inwardly directed cotransporters, NKCC-1, NKCC-2, and NCC-1, only NKCC-1 was detected at significant levels in brain. NKCC-1 was expressed in neurons, appearing first in cortical plate but not in ventricular or subventricular zone. Expression levels peaked by the third postnatal week and were maintained into adulthood. The outwardly directed cotransporters, KCC-1 and KCC-2, demonstrated significantly different levels and time courses of expression. KCC-1 was expressed prenatally at very low levels which increased little over the course of development. In contrast, KCC-2 expression appeared perinatally and increased dramatically after the first week of postnatal life. Differential changes in expression of this gene family occurred during periods of critical shifts in chloride homeostasis and GABA response suggestive of a role in these processes. Furthermore the absence of expression of known inwardly directed cotransporters in Cl- accumulating neuroepithelia and lack of evidence for glial expression suggests that as yet unidentified members of this gene family may be involved in chloride homeostasis in immature neuronal precursors and neuroglia.

Animals↗
PubMed1998

N-Arylalkyl pseudopeptide inhibitors of farnesyltransferase.

Inhibitors of Ras protein farnesyltransferase are described which are reduced pseudopeptides related to the C-terminal tetrapeptide of the Ras protein that signals farnesylation. Reduction of the carbonyl groups linking the first three residues of the tetrapeptide leads to active inhibitors which are chemically unstable. Stability can be restored by alkylating the central amine of the tetrapeptide. Studies of the SAR of these alkylated pseudopeptides with concomitant modification of the side chain of the third residue led to 2(S)-(2(S)-¿[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)- methylpentyl]naphthalen-1-ylmethylamino¿acetylamino)-4 -methylsulfany lbutyric acid (11), a subnanomolar inhibitor. The methyl ester (10) of this compound exhibited submicromolar activity in the processing assay and selectively inhibited anchorage-independent growth of Rat1 cells transformed by v-ras at 2.5-5 microM.

3T3 Cells↗
PubMed1998

Developmental expression of C1C-2 in the rat nervous system.

Regulation of expression of the voltage-gated chloride channel, C1C-2, was investigated during development and adult life in rat brain. RNase protection assays demonstrated a marked increase in levels of expression of C1C-2 in brain during early postnatal development which was also detected in adult brain. In situ hybridization of E15 and E18 rat brains demonstrated C1C-2 expression in deep brain nuclei and scattered cells within the neuroepithelial layers, but not in the regions of subventricular zone that primarily give rise to glial populations. By E18 all neurons within the emerging cortical plate and its equivalent in other areas of the CNS were heavily labeled. During the first postnatal week, C1C-2 was highly expressed in most neurons. By P7 a pattern of differential expression emerged with evidence of decreased expression of C1C-2 mRNA in many neuronal populations. In adult rat brain, C1C-2 was expressed at highest levels in large neurons as found within layer V of cortex, Ammon's Horn of hippocampus, or mitral cells of the olfactory bulb and Purkinje cells within the cerebellum. Many smaller neurons within the diencephalon maintained significant levels of expression. A functional conductance was readily detected in hippocampal neurons during the first postnatal week, which had the same characteristic properties as the conductance observed in adult neurons. The observed expression and functional presence of C1C-2 suggest a widespread role in neuronal chloride homeostasis in early postnatal life, and demonstrated that cell specific shut-down resulted in the adult pattern of expression.

Animals↗
PubMed1998

Effects of polyethylene particles on tissue surrounding knee arthroplasties in rabbits.

Clinical studies suggest a role for polyethylene (PE) wear debris in the pathogenesis of osteolysis and loosening of total joint replacements. In this study, submicron particles of ultrahigh molecular weight PE (UHMWPE) were placed around pressfit tibial hemiarthroplasties in rabbits to determine the biological reaction. After 6 months the periprosthetic tissue was harvested and characterized biochemically by measuring the extracellular matrix macromolecules, collagen, and glycosaminoglycan (GAG) and quantifying the expression of inflammatory/osteolytic mediators [prostaglandin E2 (PGE2), hexosaminidase, transforming growth factor beta (TGF beta), and interleukins-6 and -1 (IL-6, IL-1)]. Particle exposure resulted in a decrease in levels of total extracellular matrix molecules including a 53% decrease in total GAG (p < 0.05) and a 74% decrease in total collagen (p < 0.005). Collagen content remained significantly decreased when normalized for cellularity (DNA content). Total TGF beta release exhibited a downward trend (p = 0.06) in the particle exposed group. Hexosaminidase and PGE2 levels did not show a difference between groups; however, when normalized for cellularity, PGE2 values exhibited an upward trend in the particle exposed group (p = 0.1). IL-6 was undetected by bioassay and ELISA. Previous studies emphasized that PE debris enhances the degradation of bone. The data from this in vivo model suggest that submicron UHMWPE particles may also act to inhibit biosynthetic pathways of bone and mesenchymal tissue. Decreased levels of collagen, GAG, and TGF beta expression may indicate suppression of bone formation, possibly through a downregulation of osteoblast activity.

Animals↗
PubMed1998

Agonist properties of N,N-dimethyltryptamine at serotonin 5-HT2A and 5-HT2C receptors.

Extensive behavioral and biochemical evidence suggests an agonist role at the 5-HT2A receptor, and perhaps the 5-HT2C receptor, in the mechanism of action of hallucinogenic drugs. However the published in vitro pharmacological properties of N,N-dimethyltryptamine (DMT), an hallucinogenic tryptamine analog, are not consistent with this hypothesis. We, therefore, undertook an extensive investigation into the properties of DMT at 5-HT2A and 5-HT2C receptors. In fibroblasts transfected with the 5-HT2A receptor or the 5-HT2C receptor, DMT activated the major intracellular signaling pathway (phosphoinositide hydrolysis) to an extent comparable to that produced by serotonin. Because drug efficacy changes with receptor density and cellular microenvironment, we also examined the properties of DMT in native preparations using a behavioral and biochemical approach. Rats were trained to discriminate an antagonist ketanserin from an agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in a two-lever choice paradigm. Pharmacological studies showed that responding on the DOI and ketanserin lever reflected agonist and antagonist activity at 5-HT2A receptors, and hence, was a suitable model for evaluating the in vivo functional properties of DMT. Like other 5-HT2A receptor agonists, DMT substituted fully for DOI. Intact choroid plexus was used to evaluate the agonist properties at endogenous 5-HT2C receptors; DMT was a partial agonist at 5-HT2C receptors in this native preparation. Thus, we conclude that DMT behaves as an agonist at both 5-HT2A and 5-HT2A receptors. One difference was evident in that the 5-HT2C, but not the 5-HT2A, receptor showed a profound desensitization to DMT over time. This difference is interesting in light of the recent report that the hallucinogenic activity of DMT does not tolerate in humans and suggests the 5-HT2C receptor plays a less prominent role in the action of DMT.

3T3 Cells↗
PubMed1998

Dimethylamine formation in the rat from various related amine precursors.

Dimethylamine is the immediate precursor of dimethylnitrosamine, a known potent carcinogen in a wide variety of animal species. Although small amounts of dimethylamine are ingested directly, the major dietary source is believed to be via choline and related materials. Owing to quantitative recoveries following oral administration, urinary dimethylamine levels provide good overall measures of body exposure. The oral administration of equimolar amounts (1 mmol/kg body weight) of potential amine precursors to male Wistar rats produced only small increases in urinary dimethylamine after choline (+ 11%; 0.60 +/- 0.36% dose), dimethylaminopropanol (+ 32%; 1.49 +/- 0.30% dose), dimethylaminoethyl chloride (+ 110% 5.38 +/- 1.72% dose) and trimethylamine (+ 51%; 1.6 +/- 0.80% dose) input, whereas significantly larger increases were found following trimethylamine N-oxide ingestion (+ 355%; 12.93 +/- 1.13% dose; t-test, P < 0.001). These data suggest that trimethylamine N-oxide is a major dietary source of dimethylamine, by direct conversion and not by sequential reduction (to trimethylamine) and demethylation, and that in this respect it is of greater importance, on a molar basis, than choline.

Administration, Oral↗
PubMed1998

Microbial magnesium transport: unusual transporters searching for identity.

Mg2+ is unique among biological cations because of its charge density and solution chemistry. This is abundantly reflected in its transport systems, studied primarily in Salmonella typhimurium. The constitutively expressed CorA transport system is the primary Mg2+ influx pathway for the Bacteria and the Archaea. Its structure of a large N-terminal soluble periplasmic domain with three transmembrane segments at the C-terminus is unique among membrane carriers, and its protein sequence bears no resemblance to other known proteins. The MgtE transport system can also mediate Mg2+ uptake, but whether this is its primary function is not known. MgtE also lacks homology to other known proteins. In contrast, the MgtA and MgtB Mg2+ transport systems of enteric bacteria are P-type ATPases by sequence homology, mediating Mg2+ influx with, rather than against, the Mg2+ electrochemical gradient. They are closely related to mammalian Ca2+-ATPases. Expression of MgtA and MgtB is under the control of the PhoPQ two-component regulatory system, important in bacterial virulence. In S. typhimurium, MgtB is encoded by a two-gene operon mgtCB; the function of the MgtC protein is unknown, and it lacks close homologues. The ligand for the PhoQ membrane sensor kinase is Mg2+ and, at decreased extracellular Mg2+ concentrations, transcription of mgtA and mgtCB are enormously induced. All three genes are also induced upon S. typhimurium invasion of epithelial or macrophage cells. Mutation of these genes has no effect on invasion efficiency, but an insertion in mgtC renders S. typhimurium essentially avirulent in the mouse. The physiological roles of the known Mg2+ transport systems are not yet completely defined. Nonetheless, the singular sequence and apparent structure of the CorA and MgtE transport proteins, the complex regulation of MgtA, MgtB and MgtC and their involvement in pathogenesis suggests that further study will be rewarding.

Adenosine Triphosphatases↗