Biomedical subjects
Publications and source records attributed to R L Smith.
1. The synthesis of [17,17-3H2]-sparteine and its oral administration has enabled the specific identification of 17-oxosparteine as a minor urinary metabolite (approximately 1% dose) in two healthy male volunteers.
This report presents a method for analyzing matrix metalloproteinase-9 (92 kD gelatinase) MMP-9 mRNA expression using reverse transcription polymerase chain reaction (RT-PCR). With this method, chondrocytes isolated from small samples of osteoarthritic cartilage showed significantly elevated signal for MMP-9 mRNA compared to normal cartilage. In addition, cells of synovial fluid samples aspirated from osteoarthritic joints also exhibited MMP-9 expression using this technique. RT-PCR proved to be a sensitive method for assessing MMP-9 regulation in osteoarthritic and normal cartilage, and may provide a useful index of arthritic cartilage and synovial fluid cell metabolism when limited tissue is available.
Titanium alloy particles were isolated from membranes obtained at revision arthroplasty. Addition of these retrieved particles to human monocytes/macrophages in cell culture resulted in morphologic change and metabolic activation. Cells exposed to these particles actively phagocytized the metallic debris, resulting in an increase in cytoplasm and a polarization of ingested metal. The metabolic response of the macrophages included increased release of prostaglandin E2, interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha, and increased hexosaminidase activity. Increased release of interleukin-1 beta was maximal 6 to 12 hours after particle exposure. These data show that retrieved titanium alloy particles activate macrophages in vitro in an analogous fashion to that observed around failed arthroplasties.
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3,3',4,4'-Tetrachloroazoxybenzene (TCAOB) is a structural analog of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It is formed as a byproduct during the synthesis of industrial products such as herbicides. TCAOB is a ligand for the Ah receptor and, at much higher doses, exhibits toxicities similar to TCDD. Although the reduced in vivo toxicity of TCAOB probably reflects differences in disposition, this study characterized its absorption and disposition kinetics. Male Fischer 344 rats were administered [14C]TCAOB (3.4 or 34 mg/kg p.o., 3.4 mg/kg i.v.), and the excretion of the radiolabel was monitored over 96 hr. After the low and high dose, 35% and 30% of the [14C]TCAOB were eliminated in the urine, with 55% and 54% eliminated in the feces. At 96 hr, the adipose tissue:blood ratios of [14C]TCAOB equivalents were 8 and 26 for the low and high doses, respectively. After the intravenous dose of TCAOB, the adipose tissue:blood ratio was 21 at 96 hr. Other tissue:blood ratios were of little significance (0.06-3.2). Pharmacokinetic parameters indicate that the parent molecule is cleared from blood with an average half-life of 7 hr and an average clearance of 11 ml/min.kg. Absolute bioavailability was calculated to be approximately 9%. Urine contained a variety of dichlorolaniline conjugates, which support the importance of azo reduction in the disposition of TCAOB. When compared with TCDD, the absorption of TCAOB is greatly reduced and the elimination of metabolites greatly enhanced. Therefore, at equal molar oral doses, TCAOB would express lower levels of Ah receptor-mediated toxicity than those defined for TCDD.
OBJECTIVE: To find an optimal method to study antibodies reactive with monophosphoryl lipid A characteristic of oligoarticular arthritis in children. METHODS: ELISA using 3 different methods of immobilization were compared, in (1) HCO3 buffer, pH 9.6; (2) HCl, pH 2.0; and (3) methanol. Competitive inhibition studies were carried out to quantitate relative avidity of cross reactions with suspected autoantigens. RESULTS: Sera from healthy children reacted significantly more strongly with monophosphoryl lipid A after immobilization in acid or in methanol than in a basic buffer. Sera from children with oligoarticular arthritis reacted more strongly than normal sera with the basic buffer method and even more strongly with the methanol method, but were not distinguishable from normal sera with the acid method. Results with individual oligoarticular sera correlated from method to method, but results with normal sera did not. Collagen types I and II, cardiolipin, and denatured DNA can block the anti-monophosphoryl lipid A reactivity to varying degrees on plates prepared with basic buffer, but only collagen type I and DNA block reactivity on plates prepared with methanol. CONCLUSION: The epitope on monophosphoryl lipid A recognized by oligoarticular sera is differentially affected by the method of immobilization. The crossreactivity of the anti-monophosphoryl lipid A antibody in this disease is confirmed.
The tetratricopeptide repeat (TPR) is a 34-amino-acid degenerate sequence motif that is found in a large variety of proteins, both prokaryotic and eukaryotic. TPRs are usually found in tandem arrays of up to 16 copies. In this paper we identify a direct interaction between the TPRs of Ssn6, a general transcriptional repressor, and alpha 2, a cell-type regulator in Saccharomyces cerevisiae. Five of the Ssn6 TPRs were tested individually, and all were found to interact specifically with alpha 2. These results suggest a model for TPR-protein interactions and for the role that a tandem array of TPRs may have in mediating transcriptional repression.
Prolonged exposure of human plasma fibronectin (pFn) and its 40- and 21-kDa collagen/gelatin binding fragments (GBFs) to 280-nm irradiation decreased their affinity for gelatin and for TR-CB7, a fluorescently labeled CNBr fragment of the alpha-1 chain of type I collagen. Fluorescence polarization binding assays of TR-CB7 with pFn and the 40-kDa GBF yielded progressively higher Kd's with increased time of exposure to 280-nm light at 25 degrees C. Binding of nonirradiated and irradiated pFn and fragments to gelatin-Sepharose correlated with the polarization data, confirming diminished gelatin binding following exposure to 280-nm light. Fluorescence spectra of intrinsic tryptophans in the 21- and 40-kDa GBFs exhibited changes indicative of photoinduced conformational changes; the maximum fluorescence wavelength red-shifted from between 340 and 350 nm to 360 nm, with concomitant increases in fluorescence intensity. Exposure of 21- and 40-kDa GBFs and pFn to 280-nm light also generated approximately two, four, and six free sulfhydryl groups per molecule, respectively. No sulfhydryl release was observed in other Trp- and disulfide-containing proteins under the same conditions. We propose that the fluorescence changes as well as the changes in affinity for gelatin or the collagen fragment result from structural changes secondary to the breakage of disulfide bonds, as a consequence of energy transfer from nearby tryptophans in one or more of the Fn type I repeats in the gelatin binding region of fibronectin.
cDNA clones encoding five distinct members of the FMO family of man (FMOs 1, 2, 3, 4 and 5) were isolated by a combination of library screening and reverse transcription-polymerase chain reaction techniques. The deduced amino acid sequences of the human FMOs have 82-87% identity with their known orthologues in other mammal but only 51-57% similarity to each other. The hydropathy profiles of the proteins are very similar. From the calculated rate of evolution of FMOs (a 1% change in sequence per 6 million years) it would appear that individual members of the FMO gene family arose by duplication of a common ancestral gene some 250-300 million years ago. Each of the FMO genes was mapped by the polymerase chain reaction to the long arm of human chromosome 1. The localization of the FMO1 gene was further refined to 1q23-q25 by in situ hybridization of human metaphase chromosomes. RNase protection assays demonstrated that in man each FMO gene displays a distinct developmental and tissue-specific pattern of expression. In the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus its mRNA is abundant in both organs. FMO3 expression is essentially restricted to the liver in the adult and the mRNA is either absent, or present in low amounts, in foetal tissues. FMO4 is expressed more constitutively. Human FMO1 and FMO3 cDNAs were functionally expressed in prokaryotic and eukaryotic cells. FMO1 and FMO3, expressed in either system, displayed product stereoselectivity in their catalysis of the N-oxidation of the pro-chiral tertiary amines, N-ethyl-N-methylaniline (EMA) and pargyline. Both enzymes were stereoselective with respect to the production of the (-)-S-enantiomer of EMA N-oxide. But in the case of pargyline, the enzymes displayed opposite stereoselectivity, FMO1 producing solely the (+)-enantiomer and FMO3 predominantly the (-)-enantiomer of the N-oxide.
The urinary excretion of dimethylamine has been measured in 203 unrelated healthy volunteers (102 male) who maintained their normal diets. The results for female volunteers are the first reported in the literature. The average daily output was 17.43 +/- 11.80 mg (mean +/- S.D.) (21.21 +/- 14.78 male; 13.74 +/- 5.65 female) with values for the majority of the population lying within the 0.68-35.72 mg range. Four male outliers excreted up to 109.2 mg; these large amounts of dimethylamine were presumed to be of dietary origin. The literature pertaining to urinary levels of dimethylamine has been summarised and integrated with the present observations.
This study tested the effects of fluid-induced shear on high density monolayer cultures of adult articular chondrocytes. Fluid-induced shear (1.6 Pa) was applied by cone viscometer to normal human and bovine articular chondrocytes for periods of 24, 48, and 72 hours. At 48 and 72 hours, fluid-induced shear caused individual chondrocytes to elongate and align tangential to the direction of cone rotation. Fluid-induced shear stimulated glycosaminoglycan synthesis by 2-fold (p < 0.05) and increased the length of newly synthesized chains in human and bovine chondrocytes. In human chondrocytes, the hydrodynamic size of newly synthesized proteoglycans also was increased. After 48 hours of fluid-induced shear, the release of prostaglandin E2 from the chondrocytes was increased 10 to 20-fold. In human chondrocytes, mRNA signal levels for tissue inhibitor of metalloproteinase increased 9-fold in response to shear compared with the controls. In contrast, mRNA signal levels for the neutral metalloproteinases, collagenase, stromelysin, and 72 kD gelatinase, did not show such major changes. This study demonstrated that articular chondrocyte metabolism responds directly to physical stimulation in vitro and suggests that mechanical loading may directly influence cartilage homeostasis in vivo.
BACKGROUND: The context-sensitive half-time, rather than the terminal elimination half-life, has been proposed as a more clinically relevant measure of decreasing drug concentration after a constant infusion of a given duration. The context-sensitive half-time is derived from computer modelling using known pharmacokinetic parameters. The modelled context-sensitive half-time for a 3-h infusion of alfentanil is 50-55 min and is 3 min for remifentanil. The terminal elimination half-life is 111 min for alfentanil and 12-30 min for remifentanil. It has not been tested whether the modelled context-sensitive half-time reflects the true time for a 50% decrease in drug concentration or drug effect. METHODS: Thirty volunteers received a 3-h infusion of remifentanil or alfentanil at equieffective concentrations. Depression of minute ventilation to 7.5% ETCO2 was used as a measure of drug effect. Minute ventilation response was measured, and blood samples for drug concentration were taken during and after drug infusion. The recovery of minute ventilation (drug effect) and decrease in blood drug concentration was plotted, and the time for a 50% change was determined. RESULTS: The measured pharmacokinetic context-sensitive half-time for remifentanil after a 3-h infusion was 3.2 +/- 0.9 min, and its pharmacodynamic offset was 5.4 +/- 1.8 min. Alfentanil's measured pharmacokinetic context-sensitive half-time was 47.3 +/- 12 min, and its pharmacodynamic offset was 54.0 +/- 48 min. The terminal elimination half-life modelled from the volunteers was 11.8 +/- 5.1 min for remifentanil and 76.5 +/- 12.6 min for alfentanil. CONCLUSIONS: The measured context-sensitive half-times were in close agreement with the context-sensitive half-times previously modelled for these drugs. The results of this study confirm the value of the context-sensitive half-time in describing drug offset compared to the terminal elimination half-life.
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The adherence of Candida albicans to extracellular matrix proteins may be a critical step in the pathogenesis of candidiasis. Yeast cell adherence to type I and IV collagen, fibronectin and laminin was blocked by peptide fragments from denatured type I collagen (gelatin). Gelatin fragments were obtained by digestion of the reduced protein with trypsin or CNBr. The fragments did not have antifungal properties, presumably inhibiting adherence by blocking receptors (adhesins) on the surface of the fungus. A 10-mer (GQRGVVGLPG) fashioned from the alpha-1 chain of type I collagen reduced adherence by 68%. However, a gelatin peptide possessing 47 amino acids reduced fungal adherence to type I collagen by 100%. Peptides derived from the biocompatible protein gelatin, therefore, may have a potential role in reducing the adherence of the fungus to host proteins.
The MM281 strain of Salmonella typhimurium possesses mutations in each of its three Mg2+ transport systems, requires 100 mM Mg2+ for growth, and was used to screen a genomic library from the gram-negative bacterium Providencia stuartii for clones that could restore the ability to grow without Mg2+ supplementation. The clones obtained also conferred sensitivity to Co2+, a phenotype similar to that seen with the S. typhimurium corA Mg2+ transport gene. The sequence of the cloned P. stuartii DNA revealed the presence of a single open reading frame, which was shown to express a protein with a gel molecular mass of 37 kDa in agreement with the deduced size of 34 kDa. Despite a phenotype similar to that of corA and the close phylogenetic relationship between P. stuartii and S. typhimurium, this new putative Mg2+ transporter lacks similarity to the CorA Mg2+ transporter and is instead homologous to MgtE, a newly discovered Mg2+ transport protein from the gram-positive bacterium Bacillus firmus OF4. The distribution of mgtE in bacteria was studied by Southern blot hybridization to PCR amplification products. In contrast to the ubiquity of the corA gene, which encodes the dominant constitutive Mg2+ influx system of bacteria, mgtE has a much more limited phylogenetic distribution.