Search PubMed⌕ Search

Biomedical subjects

K E Smith

Publications and source records attributed to K E Smith.

At least 127 records · Page 7Linked to original sources
PubMed1992

The epidemiology of toxoplasmosis on Iowa swine farms with an emphasis on the roles of free-living mammals.

Multiparous sows from 19 central Iowa swine farms were tested for antibodies against Toxoplasma gondii by the modified direct agglutination test. Antibody titers of 1:32 or greater were considered positive. Rodents, domestic cats, opossums (Didelphis virginiana), raccoons (Procyon lotor) and striped skunks (Mephitis mephitis) were live-trapped on each farm and similarly tested for antibodies. The overall prevalence of T. gondii antibodies in the species tested was 39/273 (14.3%) swine, 31/74 (41.9%) cats, 2/588 (0.3%) house mice (Mus musculus), 0/21 mice of the genus Peromyscus, 0/9 Norway rats (Rattus norvegicus), 1/34 (2.9%) opossums, 4/14 (28.6%) raccoons and 2/7 (28.6%) striped skunks. The overall prevalence was significantly greater in adult cats versus juvenile cats, adult male cats versus adult female cats, and adult raccoons versus juvenile raccoons. The prevalence of T. gondii antibodies in sows was compared with the prevalence in each non-swine species on a farm basis in order to identify existing associations. The prevalence in sows (and each of the non-swine species) was also analyzed on a farm basis for association with farm characteristics or swine management practices, including the degree of confinement of swine, population size and average parity of breeding female swine, estimated cat population size, and estimated mouse and rat abundance. Average titers of seropositive animals were compared on a species basis. The prevalence in sows which were totally and continuously confined was lower than that in sows which were not totally and continuously confined. The prevalence in sows from farms with an average parity of less than 2.0 was significantly lower than that in sows from farms with an average parity of 2.0 or greater. These results suggested that the prevalence of T. gondii antibodies in swine increased with age and that prevalence in swine could be reduced through total confinement. No associations could be established between prevalence in sows and prevalence in non-swine species or other farm characteristics/swine management practices. However, the high prevalence of T. gondii antibodies in cats suggested that fecal contamination of the environment by cats may be the most significant source of toxoplasmosis for swine. The extremely low prevalence of T. gondii antibodies in house mice suggested that this species was not an important source of T. gondii for swine in Iowa.

Animals↗
PubMed1992

Cloning and expression of a glycine transporter reveal colocalization with NMDA receptors.

A complementary DNA clone encoding a transporter for glycine has been isolated from rat brain, and its functional properties have been examined in mammalian cells. The transporter displays high affinity for glycine (KM approximately 100 microM) and is dependent on external Na+ and Cl-. Northern blot analysis indicates that the distribution of the mRNA encoding the glycine transporter is restricted to the nervous system. In situ hybridization data are consistent with roles for the transporter in both glycine neurotransmission and glycine modulation of N-methyl-D-aspartate (NMDA) receptors in the hippocampus. The identification of this transporter therefore opens the study of the molecular mechanisms underlying both inhibitory glycinergic transmission and NMDA-mediated excitatory transmission.

Amino Acid Sequence↗
PubMed1992

Arginine decarboxylase of oats is clipped from a precursor into two polypeptides found in the soluble enzyme.

We have examined soluble oat (Avena sativa) arginine decarboxylase by probing its structure with polyclonal antibodies that separately recognize amino-terminal and carboxyl-terminal antigens and with a monoclonal antibody that immunoprecipitates enzyme activity. These experiments indicated that oat arginine decarboxylase is clipped from a 66,000-D precursor polypeptide into 42,000- and 24,000-D produce polypeptides. Both of these are found in the enzyme and may be held together by disulfide bonds. A full-length precursor protein could not be detected in plants but could be produced by expression of the cDNA in Escherichia coli. Analysis of the expression of the cDNA in E. coli, with antibodies and using pulse labeling with [(35)S]methionine, indicated that the bulk of the expressed protein was the full-length 66,000-D form. Small amounts of 42,000- and 24,000-D polypeptides could also be detected. A reconstruction experiment, adding a radioactively labeled full-length protein isolated from E. coli to powdered oat leaves, supported the idea that the protein extraction method used for western blots was not likely to result in artifactual proteolytic degradation.

Journal Article↗
PubMed1992

Cloning and expression of a high affinity taurine transporter from rat brain.

A cDNA clone (designated rB16a) encoding a taurine transporter has been isolated from rat brain, and its functional properties have been examined in mammalian cells. The nucleotide sequence of the clone predicts a 621-amino acid protein with significant homology to other neurotransmitter transporters. Hydropathy analysis reveals stretches of hydrophobic amino acids indicative of 12 transmembrane domains, similar to those proposed for other cloned neurotransmitter transporters. The transporter displays high affinity for taurine (Km approximately 40 microM) and is dependent on external sodium and chloride for transport activity. Specific transport is sensitive to inhibition by beta-alanine and gamma-aminobutyric acid, similar to taurine transporters in vivo. Localization studies demonstrate that the transporter mRNA is located in both the brain and peripheral tissues. The structural similarity of the taurine transporter to neurotransmitter transporters is consistent with a neuromodulatory role for taurine in the nervous system.

Amino Acid Sequence↗
PubMed1991

Microbial transformation of steroids--VII. Hydroxylation of progesterone by extracts of Phycomyces blakesleeanus.

Post mitochondrial supernatants (S-12 extracts) were prepared from Phycomyces blakesleeanus by grinding washed and frozen mycelial cakes in fine sand and extracting the paste produced with buffer containing Tris-HCl pH 7.8 (0.1 M), EDTA (0.01 M), dithiothreitol (5 mM) and glycerol (10% v/v). The S-12 extracts, obtained in this way, reproducibly hydroxylated progesterone, producing 7 alpha- and 15 beta-hydroxyprogesterone the major products of whole-cell transformation. Cell-free progesterone hydroxylation was found to be approximately linearly dependent on extract concentration, to require reduced NADP (partly replaceable by NADH), and to be dependent on progesterone (apparent Km calculated to be 4 mM). K+ and Mg2+ were found not to be required. Maximum progesterone hydroxylation occurred after 2 h at pH 7.8 and at 24 degrees C. Using optimum conditions S-12 extracts were capable of hydroxylating between 5 and 15% of added progesterone (0.2 mM). Hydroxylation was found to be partially inhibited by carbon monoxide (ca 40%) and almost completely inhibited by azoles, ketoconazole and diconazole. The NADPH and molecular oxygen requirements were replaceable by NaIO4. These findings strongly suggest that hydroxylation was being catalyzed by cytochrome P-450. This was confirmed by preparing progesterone-hydroxylating microsomes and Triton N-101-solubilized microsome extracts, and by obtaining a dithionite-reduced carbon monoxide-difference absorption spectrum peak at 455 nm in the solubilized microsome extracts.

Carbon Monoxide↗
PubMed1991

The effect of high buffer cardioplegia and secondary cardioplegia on cardiac preservation and postischemic functional recovery: a 31P NMR and functional study in Langendorff perfused pig hearts.

High buffer cardioplegia may provide protection against ischemic damage by reducing the extent of intracellular acidosis. Secondary cardioplegia may improve postischemic recovery by restoration of high energy phosphates, ionic gradients, and intracellular pH. To test these hypotheses, pig hearts were arrested with high buffer (150 mM MOPS) cardioplegia or modified St. Thomas' solution II and then kept ischemic at 12 degrees C for 8 h. High energy phosphates and intracellular pH were followed during the period of ischemia, using 31P nuclear magnetic resonance spectroscopy, and functional recovery was followed during reperfusion. The hearts arrested by high buffer cardioplegia showed significantly higher intracellular pH than hearts preserved with St. Thomas' solution, but there were no significant differences in high energy phosphates. There were no significant differences in functional recovery. We found, however, that secondary cardioplegia abolished ventricular fibrillation, and resulted in improved functional recovery after 8 h of ischemic preservation compared with the hearts reperfused with Krebs-Henseleit solution alone. Our results suggest that despite attenuating the decreases in intracellular pH, high buffer cardioplegia does not improve recovery following 8 h of preservation at 12 degrees C. Secondary cardioplegia reduces the incidence of ventricular fibrillation and improves postischemic functional recovery of the myocardium.

Adenosine Triphosphate↗
PubMed1990

The ATP binding site of the yeast plasma membrane proton-translocating ATPase.

Photoaffinity labeling of the active site of the yeast plasma membrane H(+)-ATPase has been studied with 2-azido-AMP and 2-azido-ATP. The ATPase activity of the enzyme decreases as the time of photolysis of the photoactive nucleotides in the presence of the enzyme increases. The covalent incorporation of [alpha-32P]2-azido-AMP into the enzyme and the inhibition of ATPase activity have comparable time courses. ATP protects the ATPase from incorporation of and photoinactivation by 2-azido-ATP or 2-azido-AMP. In the dark, 2-azido-ATP inhibits the ATPase at concentrations comparable to the apparent Michaelis constant for MgATP. After photolysis and proteolysis of the protein, three overlapping peptides labeled by the nucleotide analogues were purified by reversed-phase high performance liquid chromatography and sequenced. The peptides are derived from a region of the ATPase that is highly conserved in related cation pumps forming a phosphorylated intermediate during the catalytic cycle. Labeling with both nucleotide analogues occurs in peptides containing residues from aspartate 560 to lysine 566. The amino acids in this region conform to a consensus sequence for ATP binding derived from phosphofructokinase.

Adenosine Triphosphate↗
PubMed1990

Microbial transformations of steroids--VI. Transformation of testosterone and androstenedione by Botryosphaerica obtusa.

The 7 beta progesterone-hydroxylating microorganism Botryosphaerica obtusa was tested for its ability to hydroxylate at this site the C-19 androstene-based compounds, androstenedione (androst-4-ene-3,17-dione) and testosterone (17 beta-hydroxyandrost-4-en-3-one). Only very limited 7 beta hydroxylation of both substrates was observed. The products included traces of 7 beta-monohydroxytestosterone and 6 beta,7 beta-dihydroxyandrostenedione from testosterone, and of 6 beta,7 beta-dihydroxyandrostenedione from androstenedione. 6 beta,7 beta-Dihydroxyandrostenedione does not appear to have been reported previously as a microbial transformation product. Both substrates were monohydroxylated in significant amounts at the isomeric 7 alpha site and at the 6 beta site. Testosterone was also significantly monohydroxylated at the 15 alpha site and in minor amounts at the 11 alpha and 12 beta sites. Some monohydroxytestosterones had also been oxidised at their 17-OH group, converting them into the corresponding monohydroxy androstenediones. The 7 alpha-hydroxy metabolites and 15 alpha-hydroxytestosterone being chemically demanding to synthesis are valuable microbial transformation products.

Androstenedione↗
PubMed1989

Autonomic and myocardial changes in middle cerebral artery occlusion: stroke models in the rat.

Stroke models in larger animals such as the cat, dog and monkey are becoming increasingly more expensive and less readily available. However, the rat is an excellent model for focal cerebral ischemia. Rats are readily available, inexpensive and their neuroanatomy and brain function have been studied extensively. Increases in plasma catecholamines and myocardial damage have been observed in clinical stroke. We examined autonomic and myocardial changes in two rat stroke models. In one model only the middle cerebral artery was occluded (MCAO) while the other model involved occlusion of both the MCA and the common carotid artery (MCAO/CCAO). Arterial blood pressure and heart rate were monitored continuously in 25 male rats (326-430 g) that underwent one of the following procedures: (1) MCAO only; (2) MCAO/CCAO; (3) CCAO only; and (4) sham occlusions (SHAM). Arterial blood samples (0.5 ml) for radioenzymatic assay of norepinephrine (NE) and epinephrine (E) were taken twice before the occlusions and at 90 and 180 min after the occlusions. The animals were perfused at the end of the experiment and the heart removed and examined histologically. Tetrazolium salts were reacted with oxidative enzymes to delineate the region of inadequate perfusion. The mean blood pressure and pulse pressure of the SHAM, MCAO/CCAO and CCAO groups significantly declined from initial values (from an average of 78 to 53 mm Hg) during the course of the experiment. However, the mean blood pressure and pulse pressure of the MCAO rats did not change during the experiment, so that the final mean blood pressure and pulse pressure were significantly higher than in the other 3 groups. The levels of both NE and E increased significantly (NE, 1443 +/- 285.9 to 4095 +/- 929 pg/ml; E, 2402 +/- 623 to 3741 +/- 1166 pg/ml) following occlusion in the MCAO group only while the other 3 groups did not change. Four of 6 hearts in the MCAO group were abnormal, showing evidence of subendocardial hemorrhage, ischemic damage or subendocardial congestion. MCAO also resulted in a consistent region of the brain with inadequate perfusion including the insular cortex. These autonomic and myocardial changes appear to mimic some of the changes seen clinically in stroke patients and provide the first acute stroke model for studying autonomic dysfunction in the rat.

Animals↗
PubMed1989

Microbial transformation of steroids--II. Transformations of progesterone, testosterone and androstenedione by Phycomyces blakesleeanus.

Phycomyces blakesleeanus transformed progesterone, testosterone and androstenedione into mixtures of products. Five monohydroxylated metabolites were obtained in reasonable yields from the progesterone transformation. Only 7 alpha- and 15 beta-hydroxyprogesterone have been reported previously from this organism. We find that it gives these two metabolites and also 6 beta-, 14 alpha- and 15 alpha-hydroxyprogesterone as major products. Five compounds were also purified from testosterone transformation mixtures. Two of these were monohydroxylated, two were ring A dehydrogenation products, and two were oxidised at C-17. The products were identified as 6 beta-hydroxytestosterone, 7 alpha-hydroxytestosterone, androsta-1,4-diene-3,17-dione (1-dehydroandrostenedione), 17 beta-hydroxyandrosta-1,4-diene-3-one (1-dehydrotestosterone) and androstenedione. All five metabolites were produced in reasonable yields, although hydroxylation was the minor transformation in this case. Only two significant products were formed from androstenedione. Both were reduced at C-17; one was also monohydroxylated. They were testosterone and 14 alpha-hydroxytestosterone. The testosterone and androstenedione transformation products have not been reported previously for this organism. We also report for the first time the preparation of P. blakesleeanus cell-free extracts which transformed progesterone reasonably efficiently and faithfully in vitro, although the proportions of each product varied from one extract to another.

Androstadienes↗
PubMed1989

Microbial transformations of steroids--V. Transformation of progesterone by whole cells and extracts of Botryosphaerica obtusa.

Members of the genus Botryosphaerica are reported 7 alpha steroid hydroxylators [1]. We found that the species B. obtusa efficiently hydroxylated progesterone in a 1-day transformation but it gave 7 beta-hydroxyprogesterone as the main product rather than the expected 7 alpha-hydroxy isomer, which was produced in only trace amounts. Also formed in minor amounts were 6 beta-, possibly 9 alpha- (see main text), 14 alpha- and 15 beta-monohydroxyprogesterones. The transformation mixtures included appreciable amounts of dihydroxylated progesterones which were mainly based on 7 beta-hydroxyprogesterone. The second hydroxyl group was at one of the minor monohydroxylation sites. The relative concentrations of the progesterone diols increased and those of the mono-alcohols concomitantly decreased when transformation was extended beyond 1 day. Monohydroxylated 6-dehydroprogesterones began to accumulate after about 3 days and these compounds seemed to have been formed by 6,7-dehydration of the dihydroxyprogesterones. We prepared mycelial cell-free extracts which were capable of transforming progesterone and retained the site-specificity of whole cells. These extracts converted 7 beta-hydroxyprogesterone to its 6-dehydro derivative, confirming that ring B desaturation occurs in this organism by dehydration. The dehydratase activity necessary for the conversion was separable from the hydroxylase activity by ultra-centrifugation. All hydroxylase activity co-sedimented with the membrane fraction, implying that steroid hydroxylation is effected by a membrane-bound enzyme(s). Dehydratase activity was present in both the pellet and the supernatant fractions, which suggests that it may involve a loosely bound, and easily removed, membrane-associated enzyme.

Biotransformation↗
PubMed1989

Microbial transformations of steroids--IV. 6,7-Dehydrogenation; a new class of fungal steroid transformation product.

Microbial steroid dehydrogenation is quite common. The reaction seems to occur mainly in bacteria and usually results in hydrogen abstraction from positions C(1)-C(2) and/or C(4)-C(5) with occasional aromatisation of ring A. We have screened large numbers of fungal cultures for their ability to monohydroxylate steroids at unusual sites and in the course of our investigations we have identified seven fungal strains capable of dehydrogenating ring B of progesterone and androstenedione at positions C(6)-C(7). Microbiological dehydrogenation at this site seems not to have been reported previously. The structures of the metabolites isolated from progesterone, and the producing fungi, are: 6-dehydroprogesterone (Botryodiplodia theobromae), 11 alpha-hydroxy-6-dehydroprogesterone (Botryosphaerica obtusa, Mucor racemosus and Nigrospora sphaerica), 12 alpha-, 15 beta- and 16 alpha-hydroxy-6-dehydroprogesterones (B. obtusa) and 14 alpha-hydroxy-6-dehydroprogesterone (Apiocrea chrysosperma) [1]. From androstenedione we isolated 6-dehydroandrostenedione (Absidia coerulea and Curvularia lunata) and 6-dehydrotestosterone (C. lunata).

Androstenedione↗
PubMed1989

Microbial transformations of steroids--III. Transformation of progesterone by Sepedonium ampullosporum.

The 16 alpha-steroid hydroxylating fungus Sepedonium ampullosporum (CMI strain 203 033) transformed progesterone into 16 alpha-hydroxyprogesterone and four other major metabolites which have not been reported previously for this organism, 6 beta-hydroxyprogesterone, 17 alpha-hydroxyprogesterone, 16 alpha-hydroxyandrostenedione and 16-oxotestosterone (16-ketotestosterone). Among the minor metabolites we have been able to identify 15 alpha-hydroxyprogesterone. This compound has not been reported for S. ampullosporum. The conditions used for transformation had comparatively little effect on the relative proportions of products formed, 16 alpha-hydroxyprogesterone always being the predominant metabolite, but had a major effect on the total yields of metabolites isolatable. These findings suggest that one or more constitutive enzyme systems were responsible for the transformations.

Biotransformation↗
PubMed1989

Resolution and purification of a neurofilament-specific kinase.

Both in vivo and in vitro, neurofilaments (NFs) are among the most highly phosphorylated proteins known. The majority of the NF phosphorylation sites reside on the carboxyl-terminal tails of the proteins. We have isolated and characterized an effector-independent neurofilament-specific protein kinase from bovine spinal cord that is associated with the NF complex and exhibits a marked substrate specificity for NF-H, the largest subunit of the NF triplet. This kinase activity emerges from a NF-conjugated affinity column coincident with a 67-kDa doublet on NaDodSO4/polyacrylamide gels and has a purity of greater than 90%. The purified enzyme exclusively phosphorylates NF-H tails and is dependent on prior phosphorylation of this molecule. The enzyme is also not autophosphorylated. While the molecular properties and substrate specificities of the NF kinase distinguish it from cAMP-dependent protein kinase, protein kinase C, Ca2+/calmodulin kinase, and casein kinases I and II, it exhibits certain properties similar to, but different from, the growth-associated histone H1 kinase. The molecular properties and specific sequence requirements of the NF kinase suggest that this enzyme could play a pivotal role in the phosphorylation of NFs in normal and pathological states such as Alzheimer disease, where NFs are hyperphosphorylated.

Animals↗
PubMed1989

Assembly and exchange of intermediate filament proteins of neurons: neurofilaments are dynamic structures.

We have explored the dynamics of intermediate filament assembly and subunit exchange using fluorescently labeled neurofilament proteins and a fluorescence resonance energy transfer assay. Neurofilaments (NFs) are assembled from three highly phosphorylated proteins with molecular masses of 180 (NF-H), 130 (NF-M), and 66 kD (NF-L) of which NF-L forms the structural core. The core component, NF-L, was stoichiometrically labeled at cysteine 321 with fluorescein, coumarin, or biotin-maleimide to produce assembly-competent fluorescent or biotinylated derivatives, respectively. Using coumarin-labeled NF-L as fluorescence donor and fluorescein-labeled NF-L as the fluorescence acceptor, assembly of NF filaments was induced by rapidly raising the NaCl concentration to 170 mM, and the kinetics was followed by the decrease in the donor fluorescence. Assembly of NF-L subunits into filaments does not require nucleotide binding or hydrolysis but is strongly dependent on ionic strength, pH, and temperature. The critical concentration of NF-L, that concentration that remains unassembled at equilibrium with fully formed filaments, is 38 micrograms/ml or 0.6 microM. Under physiological salt conditions NF-L filaments also undergo extensive subunit exchange. Kinetic analysis and evaluation of several possible mechanisms indicate that subunit exchange is preceded by dissociation of subunits from the filament and generation of a kinetically active pool of soluble subunits. Given the concentration of NF-L found in nerve cells and the possibility of regulating this pool, these results provide the first information that intermediate filaments are dynamic structures and that NF-L within the NF complex is in dynamic equilibrium with a small but kinetically active pool of unassembled NF-L units.

Animals↗
PubMed1989

Reducing distress during invasive medical procedures: relating behavioral interventions to preferred coping style in pediatric cancer patients.

The hypothesis that matching childrens' preferred coping styles (repression, sensitization) with behavioral interventions (distraction, sensory information) during invasive medical procedures will reduce self-reports of fear and pain, anticipatory heart rate, and observable signs of distress was tested in 28 children with cancer. A significant coping style by intervention interaction for the self-report of experienced pain was found. However, those groups using an intervention that matched their preferred coping style actually reported higher experienced pain ratings. Trends for differences between coping style on factors indicative of the chronicity of the disease were found. Chronicity, which reflects the degree of past experience with invasive medical procedures, may be a more important factor than preferred coping style in pain management of these patients.

Adaptation, Psychological↗
PubMed1989

Serum antibody responses to the N-acetylneuraminyllactose-binding hemagglutinin of Campylobacter pylori.

We recently reported that Campylobacter pylori possesses a surface-associated fibrillar hemagglutinin which has an affinity for N-acetylneuraminyllactose; this hemagglutinin may function as a colonization factor for attachment to the gastric epithelium. In the present study we examined serum samples obtained from 65 C. pylori-infected individuals with gastric ulcers, duodenal ulcers, or both and from 121 asymptomatic volunteers, including 62 who were infected with C. pylori, for immunoglobulin G (IgG) specific for the C. pylori neuramin-lactose-binding hemagglutinin (NLBH). The NLBH purified from a single isolate was used as the antigen in an enzyme-linked immunosorbent assay. A positive result was obtained with serum from 81.5% (53 of 65) of the individuals with ulcers, 67.7% (42 of 62) of the C. pylori-infected volunteers, and 1.7% (1 of 59) of the C. pylori-negative volunteers. This result confirms that NLBH is produced in vivo. There are several possible explanations for the fact that only 75% of 127 C. pylori-infected individuals were positive for anti-NLBH IgG in serum. We favor the hypothesis that there are probably several antigenically distinct C. pylori NLBHs, i.e., NLBHs which would stimulate antibody that was undetectable by the antigen used in the enzyme-linked immunosorbent assay described here.

Antibodies, Bacterial↗