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Biomedical subjects

R L Smith

Publications and source records attributed to R L Smith.

At least 199 records · Page 11Linked to original sources
PubMed1995

Cloning and characterization of MgtE, a putative new class of Mg2+ transporter from Bacillus firmus OF4.

The MM281 strain of Salmonella typhimurium which possesses mutations in each its three known Mg2+ transport systems and requires 100 mM Mg2+ for growth was used to screen a genomic library from the gram-positive alkaliphilic bacterium Bacillus firmus OF4 for clones that could restore the ability to grow without Mg2+ supplementation. Of the clones obtained, five also conferred sensitivity to Co2+, similar to the phenotype of mutants with mutations in the S. typhimurium corA Mg2+ transport locus. All five contained identical inserts by restriction analysis. Using 63Ni2+ as a surrogate for the unavailable 28Mg2+, the plasmid insert was shown to restore cation uptake with properties similar but not identical to those of the S. typhimurium CorA Mg2+ transporter. Sequence analysis of one clone identified a single open reading frame with multiple possible initiation sites. Deletion and mutation analysis identified a minimum open reading frame of 939 bp encoding a polypeptide with a predicted molecular mass of 34 kDa. Disruption of the open reading frame eliminated cation influx activity and restored resistance to Co2+. This putative transporter, designated MgtE, has no sequence similarity to any known protein including CorA and appears to represent a new class of Mg2+ transport system.

Amino Acid Sequence↗
PubMed1995

Distribution of the CorA Mg2+ transport system in gram-negative bacteria.

The CorA Mg2+ transport system is the dominant constitutive uptake mechanism in Salmonella typhimurium and Escherichia coli. Southern blot hybridization and PCR techniques were used to screen a panel of 18 additional gram-negative bacterial species for corA homologs. Virtually all strains tested positive for the presence of corA. Thus, corA appears to be ubiquitous within gram-negative bacteria and is likely their major Mg2+ influx system.

Bacterial Proteins↗
PubMed1995

Long-term expression in sensory neurons in tissue culture from herpes simplex virus type 1 (HSV-1) promoters in an HSV-1-derived vector.

Amplicons, defective herpes simplex virus type 1 (HSV-1) vectors, were constructed to use four HSV-1 promoters, from the immediate-early (IE) 1 IE 3, IE 4/5, and late glycoprotein C (gC) genes, to regulate expression of the Escherichia coli lacZ gene, encoding beta-galactosidase, and packaged into infectious particles. Infection of sensory neurons in vitro with amplicons containing the IE 1, IE 3, or IE 4/5 promoter resulted in stable long-term expression of beta-galactosidase from 2 to 10 weeks after gene transfer. The number of neurons expressing beta-galactosidase was not changed by treatments previously shown to produce reactivation of latent HSV-1. In addition, the latency-associated transcript was detected in many of the same neurons that expressed beta-galactosidase, indicating that the viral IE promoters in the amplicons can function in the same neurons that harbor latent virus. Delivery of beta-galactosidase protein directly into neurons by microinjection indicated that the half-life for histochemical detection of beta-galactosidase was between 24 and 48 h, suggesting that the persistence of beta-galactosidase histochemical staining cannot be explained by the stability of the reporter protein alone. In contrast to the IE promoters, the gC promoter of the late gene class did not support long-term expression of beta-galactosidase; instead, beta-galactosidase was detected in only a few neurons per culture at 2 weeks after infection, and superinfection with wild-type HSV-1 did not increase the level of expression from the gC promoter. These results suggest that the HSV-1 IE promoters in the amplicons are not subject to the promoter inactivation that occurs with many types of virus vectors and that the IE promoters in the context of the amplicon avoid the promoter inactivation observed from the same promoters in the HSV-1 genome during latency.

Animals↗
PubMed1995

Gap junctions between mast cells and fibroblasts in the developing avian eye.

Mast cells are present in the eye of chick embryos from the 14th day onward, displaying metachromatic granules, mainly in the iris anterior surface and pectinate ligament. Ultrastructurally these cells show electron-dense granules and a few thin and short cytoplasmic projections in close contact with fibroblasts. Sometimes these contacts are extensive, with long fibroblast projections partially involving the mast cells. Gap junctions between mast cells and fibroblasts are observed only in the eyes of 16- and 20-day-old embryos. These intercellular specializations are represented by a close apposition of cytoplasmic membranes with an extension up to 300 nm. Gap junctions between mast cells and fibroblasts were not observed previously in vivo or in vitro, although in vitro studies have shown that a number of functionally critical interactions may occur between these cells. Our morphological findings suggest that, in vivo, fibroblasts interact with mast cells and may influence their maturation.

Animals↗
PubMed1995

Interleukin-1 and tumor necrosis factor-alpha increase insulin-like growth factor-binding protein-3 (IGFBP-3) production and IGFBP-3 protease activity in human articular chondrocytes.

IGF-I is the major anabolic factor for cartilage matrix production. Chondrocytes and cartilage treated with interleukin-1 alpha (IL-1 alpha), and chondrocytes from several models of inflammatory joint disease, exhibit reduced responsiveness to IGF-I. Since the IGF-binding proteins (IGFBPs) modulate the effects of IGF-I, we examined the effect of IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) on IGFBP production by normal human articular chondrocytes in primary culture. Western ligand blots and immunoprecipitation of conditioned medium samples showed that articular chondrocytes produced IGFBPs-2, -3 and -4 and glycosylated IGFBP-4. Both IL-1 alpha and TNF-alpha increased chondrocyte production of IGFBP-3, but did not alter IGFBP-4 production. The activity of a neutral metalloprotease with the ability to cleave IGFBP-3 was also increased by IL-1 alpha. These data suggest that the cytokines IL-1 alpha and TNF-alpha may act to reduce IGF-I access to chondrocytes by increasing production of IGFBP-3. This may be a factor in the decreased matrix production in the inflammatory arthritides.

Blotting, Western↗
PubMed1995

Isolation and characterization of wear particles generated in patients who have had failure of a hip arthroplasty without cement.

Wear particles from thirty-five membranes obtained during revision hip-replacement operations were studied after digestion of the soft tissue with papain. The particles were isolated and were characterized with use of light and scanning electron microscopic techniques, x-ray microanalysis, and an automated particle analyzer. The mean size of the polyethylene particles was 0.5 micrometer, and the metal particles were a mean of 0.7 micrometer, as determined with scanning electron microscopy. The automated particle analyzer revealed a mean particle diameter of 0.63 micrometer (more than 90 per cent of all particles were less than 0.95 micrometer) and a mean of 1.7 billion particles per gram of tissue, compared with only 143 million per gram of tissue for the control samples. X-ray microanalysis revealed metal debris in sixteen (46 per cent) of the thirty-five membranes after digestion. Thirteen (50 per cent) of the twenty-six membranes surrounding a titanium-alloy stem contained metal particles, compared with three of the nine membranes surrounding a chromium-cobalt stem. Metal debris was present in only one of the twelve membranes surrounding a titanium-alloy stem without a porous coating, compared with twelve of the fourteen membranes surrounding a titanium-alloy stem with a porous coating. This tenfold difference in prevalence was significant (p < 0.005). On the average, the total number of particles (expressed in millions per gram of tissue) associated with the bipolar acetabular components was twice that associated with the fixed acetabular components. In addition, there was a trend toward a larger mean size of the polyethylene particles in association with the bipolar cups. Our data indicate that particulate prosthetic debris in the tissues around failed femoral components that have been inserted without cement constitutes a class of particles that are predominantly less than one micrometer in size and are present in amounts of more than one billion particles per gram of tissue. Routine histological methods did not detect this class of wear debris and led to a gross underestimation of the amount of debris in these membranes.

Adult↗
PubMed1995

Critical care transport: an evolving role in EMS.

Critical care transport (CCT). It is defined as the movement of critically ill patients from facilities where the patients' needs exceed available resources to places that meet their needs, while maintaining a specialized level of care. And it is a specialty that is becoming increasingly common in today's managed care environment--an environment that emphasizes putting people in network hospitals. It is also becoming a viable career move for paramedics wishing to upgrade their skills and education.

Critical Care↗
PubMed1995

Neurochemical and behavioral evidence that quipazine-ketanserin discrimination is mediated by serotonin2A receptor.

The purposes of this study were to determine: first, if animals could be trained to discriminate a serotonin (5-HT)2 receptor agonist from a 5-HT2 receptor antagonist; second, which 5-HT2 receptor subtype was mediating the cues; and third, the usefulness of this model for studying adaptive changes in the 5-HT2 receptor system. Rats were trained to discriminate quipazine (0.5 mg/kg) from ketanserin (1.0 mg/kg) on a variable interval-20 schedule of reinforcement. After acquisition, the quipazine and ketanserin dose-response curves were found to be orderly and reproducible. Additional 5-HT2 receptor agonists (2,5-dimethoxy-4-iodoamphetamine and MK 212) and antagonists (pizotifen, mianserin, pirenperone and MDL 100,907) were tested for generalization and found to substitute for the quipazine and ketanserin cues, respectively. In antagonist studies, MDL 100,907 potently blocked quipazine discrimination. Results of ex vivo binding studies designed to estimate occupancy of 5-HT2A and 5-HT2C receptors suggested that the training dose of ketanserin blocked only 5-HT2A receptors and not 5-HT2C receptors. The combined results from the substitution, antagonism and ex vivo receptor autoradiographic studies suggest that the discriminative stimuli of quipazine and ketanserin are mediated at least in part by the 5-HT2A receptor. Additional experiments were designed to study adaptive changes in the 5-HT2A receptor. A single large dose of quipazine produced a rebound ketanserin-like effect at 20 hr after administration; however, a single large dose of ketanserin (10 mg/kg) did not produce a rebound quipazine-like effect.

Animals↗
PubMed1995

The medical director in hospital-based transitional care units.

This article begins with a brief overview of what subacute care is and why it is growing exponentially. It then discusses the characteristics of patients appropriate for treatment in a transitional care unit (TCU) and the evolving role of physicians in their care. The process of care in a typical hospital-based TCU from admission to discharge is discussed with an emphasis on documentation and an interdisciplinary approach. The role of the medical director is emphasized. The article closes with strategies the authors feel are useful for improving care in hospital-based TCUs.

Aged↗
PubMed1995

Nonsteroidal antiinflammatory drugs: effects on normal and interleukin 1 treated human articular chondrocyte metabolism in vitro.

OBJECTIVE: To test the effects of nonsteroidal antiinflammatory drugs (NSAID), naproxen, ibuprofen and diclofenac, and salicylates on normal and interleukin 1 (IL-1) treated human articular chondrocyte metabolism in vitro. METHODS: Normal adult human articular chondrocytes were isolated and cultured as primary monolayers; the cells were treated with NSAID and salicylates at low and high plating density for assessing effects on proliferation and matrix synthesis and IL-1 modulated cell metabolism, respectively. RESULTS: Cell proliferation was inhibited by ibuprofen and high doses of salicylates. Glycosaminoglycan (GAG) synthesis was stimulated by ibuprofen at 10 micrograms/ml but was not changed by any other drugs at similarly low concentrations; at medium to high concentrations, only the salicylates inhibited GAG synthesis. Collagen synthesis was unaffected by any drug at the concentrations tested. IL-1 induced prostaglandin E2 release was completely inhibited by the NSAID and partially inhibited by the salicylates. IL-1 induced IL-6 release was inhibited by ibuprofen and the salicylates where as IL-1 induced APMA-activated collagenase was only inhibited by the salicylates. CONCLUSION: Normal human chondrocytes respond differentially to naproxen, ibuprofen, dicolfenac, and the salicylates; these observations suggest that each drug may exhibit unique attributes with respect to longterm efficacy on cartilage metabolism.

Cartilage, Articular↗
PubMed1995

Differential expression of an inwardly rectifying chloride conductance in rat brain neurons: a potential mechanism for cell-specific modulation of postsynaptic inhibition.

A voltage-sensitive inwardly rectifying chloride (Cl-) conductance (GCl(V) is present in hippocampal pyramidal but not dentate gyrus neurons and has a significant role in modulation of neuronal inhibition by GABA. GCl(V) has the same activation properties as the cloned and expressed Cl- channel CIC-2. In brain, CIC-2 was detected selectively in neurons, and in hippocampus was detected in the same populations of neurons that demonstrate GCl(V). CIC-2 mRNA expression varied widely in different neuronal populations in brain but was greatest in pyramidal and other large neurons and least in interneurons. The observed differential expression of CIC-2 provides a potential molecular basis for the paradoxical excitation produced by GABAA receptor activation in selected neuronal populations.

Animals↗
PubMed1994

Phosphate-regulated inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81.

A complex consisting of the cyclin-dependent kinase (CDK) PHO85 and the cyclin PHO80 phosphorylates and is thought to inactivate the transcription factor PHO4 when yeast cells are grown in medium containing high concentrations of phosphate. The CDK inhibitor PHO81 inhibits the kinase activity of the PHO80-PHO85 complex when Saccharomyces cerevisiae cells are grown in medium depleted of phosphate. A region of PHO81 with similarity to the mammalian CDK inhibitor p16INK4 is sufficient for inhibition in vitro. These studies demonstrate that CDK inhibitors are used to regulate kinases involved in processes other than cell cycle control and suggest that the ankyrin repeat motif may be commonly used for interaction with cyclin-CDK complexes.

Amino Acid Sequence↗
PubMed1994

3-substituted thieno[2,3-b][1,4]thiazine-6-sulfonamides. A novel class of topically active carbonic anhydrase inhibitors.

3-Aminoalkyl derivatives of thieno[2,3-b][1,4]thiazine-6-sulfonamide were prepared for evaluation as topically active ocular hypotensive agents. The compounds described were found to be excellent in vitro inhibitors of carbonic anhydrase II and in vivo to lower intraocular pressure in three rabbit models of ocular hypertension. Compounds 20A, 20B, and 20C met the requirement of formulation as a 1% solution at pH 5.2, but none of the compounds described exhibited greater activity in the normotensive albino rabbit, the alpha-chymotrypsin-treated albino rabbit, or the normotensive pigmented rabbit than MK-927 or MK-507, the present clinical candidates.

Administration, Topical↗
PubMed1994

Regulation of the herpes simplex virus latency-associated transcripts during establishment of latency in sensory neurons in vitro.

The temporal appearance of the major latency-associated transcript (LAT) of herpes simplex virus, type-1 (HSV-1) was examined in sensory neuronal cultures during the establishment of either a latent or a lytic infection. Under conditions that result in the establishment of a latent infection, a significant delay in LAT accumulation was observed. The delay in the appearance of LAT was reflected in both a reduced number of LAT-positive neurons detected by in situ hybridizations and by low levels of the major 2-kb LAT detected by Northern blot analysis at early times compared to later in the latent infection. The percentage of LAT-positive neurons shown by in situ hybridizations and the relative abundance of the major LAT by Northern blot analysis increased markedly by 14 days after inoculation with virus. In addition to the major LAT, a spliced 1.5-kb LAT species was detected in Northern blot analysis after establishment of latency in the neuronal cultures, similar to observations in vivo. In contrast to the latent infection, under conditions that produced lytic infections in the neuronal cultures, LAT and HSV-1-specific antigens were detected in the majority of neurons 24 hr after inoculation with virus. These results indicate that LAT expression during the establishment of latency is regulated differently than during the lytic infection: LAT expression appears to be inhibited initially during the establishment of latency, whereas LAT is readily expressed during the lytic infection.

Animals↗
PubMed1994

Drug discrimination is a continuous rather than a quantal process following training on a VI-TO schedule of reinforcement.

Debate continues as to whether drug discrimination in animals is an inherently quantal or continuous process. This issue is important in determining the appropriate interpretation of results from drug discrimination studies designed to assess the nature of drug-induced interoceptive cues. The quantal approach holds that subjects perceive a drug cue in an all-or-none manner, while the continuous view proposes that when appropriate training and testing procedures are used, subjects can discriminate along a continuum of interoceptive cues. Data consistent with the quantal view have consistently been generated by animals trained to respond on schedules of reinforcement having an FR component. Since quantal responding is a characteristic of these schedules, results from drug discrimination studies using training schedules with FR components are of little value in empirically determining whether drug discrimination reflects a quantal or continuous process. Use of variable schedules of reinforcement might be more appropriate because the pattern of responding generated does not preclude results consistent with either of the competing views. Data from the following studies that trained subjects using VI schedules with a concurrent TO for incorrect lever responding were analyzed: Barrett et al. (1982): L-5-hydroxytryptophan versus saline; Smith (1990): diazepam versus pentylenetetrazol; Barrett et al. (1992): amphetamine versus haloperidol; Barrett and Steranka (1983): amphetamine versus haloperidol. In every case, when experimental conditions produced a group mean intermediate to that for the training drugs, the distribution of scores for individual animals was normally rather than bimodally distributed.

5-Hydroxytryptophan↗
PubMed1994

Discriminative stimulus properties of the benzodiazepine receptor inverse agonist methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM).

The purpose of this study was to determine whether rats could be trained to discriminate the stimulus properties of the benzodiazepine (BZ) receptor inverse agonist DMCM from saline in a conditioned taste aversion paradigm. On a drug trial, water-deprived rats were injected with DMCM (0.55-0.6 mg/kg IP), allowed access to a 0.25% saccharin solution for 30 min, and then injected with LiCl. On non-drug trials, saline injections bracketed the drinking period. Conditioned controls were treated similarly with DMCM and saline on drug and non-drug trials, but received injections of saline instead of LiCl. At the completion of training, CMCM produced a 69% reduction of saccharin consumption on drug trials, compared with 23% for conditioned controls. The stimulus properties of DMCM were then measured by its ability to reduce the preference for saccharin over water in a two-bottle choice test. DMCM reduced saccharin preference in rats that received discrimination training from 68% to 19%, but did not alter saccharin preference in conditioned controls. Other compounds with varying activity at BZ receptors were evaluated for their ability to substitute for the discriminative stimulus effects of DMCM. Two BZ receptor inverse agonists, beta-CCE (10-18 mg/kg) and FG 7142 (3.2-18 mg/kg), substituted completely for DMCM. Partial substitution for DMCM was shown by the BZ receptor antagonist CGS 8216 (3.2-10 mg/kg) and the non-BZ convulsant pentylenetetrazol (10-20 mg/kg). The BZ receptor agonists chlordiazepoxide (0.32-5.0 mg/kg), diazepam (0.32-10 mg/kg), and alprazolam (0.1-3.2 mg/kg) and the BZ receptor antagonist flumazenil (1.0-32 mg/kg) failed to substitute for the DMCM stimulus. Pretreatment with flumazenil (1.0 mg/kg) blocked the stimulus effects of the training dose of DMCM and produced a shift to the right of the DMCM generalization curve. The pattern of compounds that substituted for the DMCM stimulus and the blockade of that stimulus by flumazenil indicate that the stimulus properties of DMCM are associated with its effects as a BZ receptor inverse agonist.

Animals↗