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K A Smith

Publications and source records attributed to K A Smith.

At least 145 records · Page 8Linked to original sources
PubMed1991

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1.

Previously, we constructed a gene disruption in the pullulanase I gene of Bacteroides thetaiotaomicron 5482A. This mutant, designated B. thetaiotaomicron 95-1, had a lower level of pullulanase specific activity than did wild-type B. thetaiotaomicron but still exhibited a substantial amount of pullulanase activity. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an alpha(1----4)-D-glucosidic bond cleaving pullulanase which has been tentatively designated a neopullulanase. The neopullulanase (pullulanase II) is a 70-kDa soluble protein which cleaves alpha(1----4)-D-glucosidic bonds in pullulan to produce panose. The neopullulanase also cleaved alpha(1----4) bonds in amylose and in oligosaccharides of maltotriose through maltoheptaose in chain length. An alpha-glucosidase from B. thetaiotaomicron 95-1 was characterized. The alpha-glucosidase was partially purified to a preparation containing three proteins of 80, 57, and 50 kDa. Pullulan and amylose were not hydrolyzed by the alpha-glucosidase. alpha(1----4)-D-Glucosidic oligosaccharides from maltose to maltoheptaose were hydrolyzed to glucose by the alpha-glucosidase. The alpha-glucosidase also hydrolyzed alpha(1----6)-linked oligosaccharides such as panose (the product of the pullulanase II action on pullulan) and isomaltotriose.

Amylases↗
PubMed1991

Interleukin-2-triggered Raf-1 expression, phosphorylation, and associated kinase activity increase through G1 and S in CD3-stimulated primary human T cells.

To gain further insight into the role of Raf-1 in normal cell growth, c-raf-1 mRNA expression, Raf-1 protein production, and Raf-1-associated kinase activity in normal human T cells were analyzed. In contrast to the constitutive expression of Raf-1 in continuously proliferating cell lines, c-raf-1 mRNA and Raf-1 protein levels were barely detectable in freshly isolated G0 T lymphocytes. Previous work with fibroblasts has suggested that Raf-1 plays a signaling role in the G0-G1 phase transition. In T cells, triggering via the T-cell antigen receptor (TCR)-CD3 complex (TCR/CD3) resulted in an approximately fourfold increase in c-raf-1 mRNA. In addition, the promotion of G1 progression by interleukin 2 (IL-2) was associated with a 5- to 10-fold immediate/early induction of c-raf-1 mRNA, resulting in up to a 12-fold increase in Raf-1 protein expression. TCR/CD3 activation did not alter the phosphorylation state of Raf-1, whereas interleukin 2 receptor stimulation resulted in a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increasing throughout G1. These findings were complemented by assays for Raf-1-associated kinase activity which revealed a gradual accumulation of serine and threonine autokinase activity in Raf-1 immunoprecipitates during G1, which remained elevated throughout DNA replication.

Antigens, CD↗
PubMed1991

Stereotaxic placement of a ventricular catheter and reservoir for the administration of morphine sulfate.

Patients with intractable pain due to cancer present a unique challenge to both medical and nursing personnel. This case study illustrates a unique home hospice managed pain control regime that has been implemented for a terminal cancer patient with intractable pain. A ventricular catheter attached to a reservoir was stereotaxically implanted for the administration of preservative-free morphine sulfate. The presentation will include the history of intraspinal morphine, the surgical placement of the ventricular access devise, and the procedure for intraventricular morphine administration. Also, the preoperative nursing assessment and patient family education will be discussed. Education of hospice nurses in the technique of injection, postoperative pain assessment, monitoring of side effects and discharge planning will conclude the presentation.

Adult↗
PubMed1990

Distinctive chromosomal structures are formed very early in the amplification of CAD genes in Syrian hamster cells.

As visualized by in situ hybridization with fluorescence detection, newly amplified CAD genes in 10(5) cell colonies are contained in multiple copies of very large regions of DNA, each tens of megabases long. The extra DNA is usually linked to the short arm of chromosome B9, which retains CAD at its normal site. The widely spaced genes are often interspersed with new G-negative regions. Individual cells within a clone have highly variable numbers of CAD genes (range 2-15). When resistant clones are examined later, at the 10(15) cell stage, the amplified genes are usually found in much more condensed structures. We propose that, in the initial event of CAD gene amplification, much of the short arm is transferred from one B9 chromosome to another. In subsequent cell cycles this initial duplication expands rapidly through unequal but homologous sister chromatid exchanges. Relatively rare secondary events lead to more condensed structures.

Animals↗
PubMed1990

Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver--conditioned medium.

We have identified a novel growth factor, stem cell factor (SCF), for primitive hematopoietic progenitors based on its activity on bone marrow cells derived from mice treated with 5-fluorouracil. The protein was isolated from the medium conditioned by Buffalo rat liver cells. It is heavily glycosylated, with both N-linked and O-linked carbohydrate. Amino acid sequence following removal of N-terminal pyroglutamate is presented. The protein has potent synergistic activities in semisolid bone marrow cultures in conjunction with colony-stimulating factors. It is also a growth factor for mast cells. In two companion papers, we present the sequences of partial SCF cDNAs, identify SCF as a c-kit ligand, and map the SCF gene to the Sl locus of the mouse.

Amino Acid Sequence↗
PubMed1990

cAMP regulation of IL-2 receptor expression. Selective modulation of the p75 subunit.

As previous experiments have shown that IL-2 activity is abrogated by cAMP, its effect on IL-2R expression by normal and leukemic T lymphocytes was evaluated in detail. The exposure of murine or human T cells to dibutyryl cAMP or the cAMP-elevating drug, forskolin, resulted in a decrease in high affinity IL-2 binding. Equilibrium binding analyses revealed that elevation of cAMP for 4 to 5 h produced a 40 to 50% decrease in the number of detectable receptors, whereas the affinity of the IL-2R interaction remained unchanged. The effect of cAMP could be attributed to a selective effect on the 75-kDa chain of the IL-2R (p75) subunit of the 55-kDa chain of the IL-2R/p75 heterodimer. The mechanism for the decreased expression of high affinity IL-2R appears to be due to a dual effect of cAMP, which functions to both increase the rate of IL-2R internalization, and to decrease the rate of expression of new receptors. Moreover, the effect of cAMP on IL-2 binding to p75 subunits is post-transcriptional, because the steady state levels of p75 mRNA expression are not altered within a time interval that produced nearly a 50% reduction in p75 binding.

Animals↗
PubMed1990

Functional consequences of interleukin 2 receptor expression on resting human lymphocytes. Identification of a novel natural killer cell subset with high affinity receptors.

In this study, we have used radiolabeled IL-2 binding assays, Northern blot analysis, immunofluorescent flow cytometry and cell sorting, as well as proliferation and cytotoxicity assays to perform an extensive phenotypic and functional characterization of the IL-2 receptor in normal resting human peripheral blood lymphocytes. Our results indicate that almost all T cells (greater than 98%) express neither the high affinity IL-2 receptor nor the functional intermediate affinity p75 chain of the IL-2 receptor without prior activation. In contrast, most NK cells constitutively express the isolated intermediate affinity p75 IL-2 receptor. In addition, a subpopulation of NK cells, distinguished by high density expression of the NKH1 antigen, constitutively express the high affinity IL-2 receptor, in addition to an excess of the isolated intermediate affinity p75 IL-2 receptor. These NKH1bright+ cells exhibit a brisk proliferative response to IL-2, similar to that seen with antigen-activated T cells, yet do so in the absence of any known antigenic stimuli. No other resting peripheral blood lymphocyte population, including CD4+, CD8+, and CD20 cells, exhibits this property. The intermediate affinity p75 IL-2 receptor, as it exists in its isolated form on resting NK cells, does not transduce a growth signal equivalent to that seen in NK cells expressing the high affinity IL-2 receptor, despite doses of IL-2 that are known to fully saturate the isolated p75 chain. This strongly suggests that additional structural or functional components are involved in generating the proliferative response following the binding of IL-2 to the high affinity heterodimeric form of the IL-2 receptor. The constitutive expression of this functional high affinity IL-2 receptor on a small population of resting NK cells provides further evidence in support of a role for these cells in the host's early defense against viral infection or malignant transformation, before the more delayed but specific T cell response.

Antibodies, Monoclonal↗
PubMed1990

Paradoxical role of GABA in a chronic model of petit mal (absence)-like epilepsy in the rat.

Neonatal Long-Evans hooded rats were treated with AY-9944, a cholesterol biosynthesis inhibitor, every 6 days for 7 weeks to induce a permanent absence-like epileptic condition. AY-9944-treated rats averaged 50 +/- 15 generalized non-motor seizures per hour of 2-15 s duration as monitored by electrocorticography. Clinically effective anti-absence drugs were observed to reduce seizure occurrence in a dose-dependent manner. Paradoxically, GABA agonists increased seizure occurrence while GABA antagonists decreased seizure occurrence. Evaluation of the benzodiazepines, diazepam and clonazepam, in this model revealed inhibition of seizure activity by GABA-independent mechanisms. Valproic acid produced a biphasic effect suggesting a GABA-independent, antiabsence action at low doses and GABAergic augmentation of seizure occurrence at higher doses. The results of this study support the hypothesis that increased GABAergic stimulation may induce inhibitory seizures in absence epilepsy.

Animals↗
PubMed1990

Evidence that the level of the p55 component of the interleukin (IL) 2 receptor can control IL 2 responsiveness in a murine IL 3-dependent cell.

Although the role of interleukin 2 (IL 2) in mature T lymphocyte function is well documented, its effect on hematopoietic progenitor cells is less well characterized. Here we have used recombinant retroviruses to transduce and express a cDNA clone encoding the p55 component of the human IL 2 receptor (h-p55), in a murine IL 3-dependent cell line, BAF3. While the parental cells do not respond to IL 2, the h-p55-expressing cells proliferate upon treatment with recombinant IL 2 after an initial lag period. The responsiveness of individual cell clones is correlated with their level of h-p55 expression, and can be inhibited by Tac monoclonal antibody. Furthermore, growth at limiting IL 2 concentrations selects a subset of cells expressing higher h-p55 levels from a bulk population. Detailed 125I-labeled IL 2 binding analysis on the highest h-p55-expressing clone detects the presence of 200 high-affinity (KD = 25 pM) IL 2 receptors. We therefore propose that the level of h-p55 expression governs the formation of high-affinity receptors, and hence IL 2 responsiveness in BAF3 cells.

Animals↗
PubMed1990

Interleukin 2 receptor-targeted cytotoxicity. Receptor binding requirements for entry of a diphtheria toxin-related interleukin 2 fusion protein into cells.

The receptor binding requirements for entry of the NAD+ ADP-ribosyltransferase component of DAB486-IL 2 into target cells were examined. Experiments utilizing cell lines bearing either high-affinity or individual subunits of the interleukin 2 receptor (IL 2R) as well as human peripheral blood mononuclear cells with natural killer activity demonstrate that the high-affinity receptor facilitates delivery of fragment A from DAB486-IL 2 to the cytosol approximately 1000 times more efficiently than either the intermediate-(p75) or low-affinity (p55) forms of the IL 2R. We show that elongation factor 2 (EF-2) in these cells is not quantitatively or qualitatively altered indicating that the relative resistance to intoxication displayed by IL 2R variant cell lines cannot be attributed to an altered intracellular target of the hybrid toxin. We also demonstrate that an alteration in the binding of DAB486-IL 2 to the p75 subunit of the IL 2R may account for the selective cytotoxicity of DAB486-IL 2 for cells bearing the heterodimeric high-affinity IL 2R.

Amino Acid Sequence↗
PubMed1990

Interleukin-2.

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Antigen-Presenting Cells↗
PubMed1990

Are the IL-2 receptors expressed in the murine fetal thymus functional?

It is well established that the majority of murine fetal thymocytes (day 15 of gestation) express receptors for interleukin 2 (IL-2), but the functional significance of these IL-2 receptors (IL-2Rs) is not clear. In situ hybridization data show a developmentally regulated expression of IL-2 and IL-2R mRNA. IL-2 binding studies were performed on fetal thymocytes and the results show the presence of both high (kD approximately equal to 20 pM) and low (kD approximately equal to 10 nM) affinity IL-2Rs. These IL-2Rs are indeed functional: intact fetal thymic lobes (but not cell suspensions) cultured in IL-2 exhibited an in vitro proliferative response at 20 pM of IL-2, corresponding with the presence of a functional high-affinity IL-2R on fetal thymocytes. The IL-2-dependent growth was primarily observed in the IL-2R+ thymic subset, which contains the CD3-/CD4-/CD8- precursor thymocytes. Furthermore, in vitro blocking of IL-2 in intact fetal thymic lobes resulted in a reduction in the cell yield, which predominantly affected the expansion of the immature CD3-/CD4-/CD8- thymocytes. Our findings strongly support the concept that the IL-2/IL-2R pathway is responsible for the proliferation of precursor cells within the fetal thymus.

Animals↗
PubMed1990

Induction of antigen-specific human cytotoxic T cells by Toxoplasma gondii.

To further the understanding of the role of T cells in immunity to the parasite Toxoplasma gondii, antigen-specific T cell clones were generated using peripheral blood mononuclear cells from seropositive individuals. Whole parasites were used to stimulate a proliferative expansion of antigen-reactive cells, followed by limiting dilution cloning in the presence of irradiated, autologous PBMC and rIL-2. Three parasite antigen-specific T cell clones expressing the CD3+ phenotype were selected for further characterization. Phenotypic analysis with monoclonal antibodies revealed two clones reactive with CD8 (RTg1 and RTg3) while the other (RTg2) phenotyped as CD4+, CD8-. When tested in a proliferation assay using a panel of different T. gondii proteins, clone RTg1 reacted with a single large protein (Mr greater than 180,000) as well as smaller components (less than 12,000), clone RTg2 reacted with a protein of Mr = 28,000 and clone RTg3 reacted with a protein of 116,000 plus smaller components (less than 12,000). Only the 28,000 = Mr antigen recognized by RTg2 was reactive on Western blot with autologous donor antisera. All three clones produced IFN-gamma and IL-2 in varying amounts upon antigenic stimulation in the presence of irradiated APC. Moreover, one clone RTg1, exhibited direct parasite cytotoxicity, inhibiting extracellular T. gondii by greater than 70% when incubated at an effector/target ratio of 40:1. This clone was alpha, beta TCR heterodimer positive and exerted its cytotoxic parasiticidal activity in the apparent absence of MHC restriction. The results provide evidence for the existence of circulating antigen-specific cytotoxic T cells in normal humans who are toxoplasma antibody seropositive.

Animals↗
PubMed1989

Structural significance of the C-terminal amphiphilic helix of interleukin-2.

The structural significance of C-terminal amphiphilic alpha-helix of human interleukin-2 has been investigated using principles of protein design. Employing disulfide-mediated semi-synthesis, several multiple residue substitution patterns were studied in order to provide rapid insight into the most appropriate features to incorporate into fully recombinant proteins. Substitutions directed toward both stabilization and destabilization of the helix resulted in proteins with modulated bioactivity. Circular dichroism verified the conformational integrity and thermal stability of the derivatives. The biologic characteristics of each derivative were evaluated in the standard murine CTLL-2 assay and compared to activities exhibited in both human T-cell bioactivity and binding assay. A strategy for the design of protein ligand agonists and antagonists without knowledge of receptor contact residues is discussed.

Binding, Competitive↗
PubMed1989

Monoamine oxidase inhibition as a sequel of hydrogen sulfide intoxication: increases in brain catecholamine and 5-hydroxytryptamine levels.

Administration of sodium hydrosulfide (NaHS), an alkali salt of hydrogen sulfide (H2S) at doses of 10 and 30 mg/kg, corresponding to sublethal and lethal doses (0.66 and 2.0 X LD50) resulted in significant increases in regional catecholamine levels of the rat brain only after the dose of 2.0 x LD50 of NaHS. Whereas the cortex and the cerebellum showed little or no change in catecholamine content, the hippocampus, striatum and brainstem all showed increases in noradrenaline and adrenaline. Additional analysis also showed that brainstem dopamine and 5-hydroxytryptamine levels (5-HT) increased as well. In vitro testing of sulfide for inhibition of monoamine oxidase (MAO) activity showed the anion to be inhibitory with an IC50 of 39.1 +/- 3.6 microM. Inhibition of MAO activity ex vivo could be demonstrated at a dose of 100 mg/kg but not at the lower dose of 30 mg/kg NaHS. Inhibition of enzyme activity could not be demonstrated at this lower dose, possibly due to the well known rapid intramitochondrial metabolism of sulfide. Correlation of synaptosomal and mitochondrial sulfide levels with enzyme inhibition data suggests that inhibition of MAO may be an important contributing factor to the mechanism(s) underlying loss of central respiratory drive after fatal intoxication with H2S.

Animals↗