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Biomedical subjects

D A Johnson

Publications and source records attributed to D A Johnson.

At least 127 records · Page 7Linked to original sources
PubMed1995

Regulation of cAMP-dependent protein kinase: enzyme activation without dissociation.

It has become axiomatic that, in contrast to other protein kinases, cAMP-dependent protein kinase (cAPK) is activated only when its catalytic (C) and regulatory (RII2) subunits dissociate. To directly evaluate this postulation, the ability of cAMP to dissociate the holoenzyme form of cAPK was examined by measuring the rotational mobility of the carboxyfluorescein-labeled C subunit (CFC) complexed to the dimeric RII2 regulatory subunit under equilibrium conditions. The rotational mobility was determined from an analysis of the time-resolved emission anisotropy of the CFC subunit. The time-resolved anisotropy decays were best fitted by a sum of two exponentials for both the free CFC subunit and the RII2CFC2 complex (holoenzyme). In the absence of cAMP, the two rotational correlation times (phi F and phi S) were 1.7 +/- 0.3 and 18.3 +/- 0.7 ns for the free CFC subunit and 2.3 +/- 0.1 and 93 +/- 2 ns for the RII2CFC2 complex, respectively. The faster rotational correlation times can be attributed to the localized rotations of the label and the slower rotational correlation times to the global rotations of the entire molecule. The addition of cAMP had no significant effect on either the fast or the slow rotational correlation time of the RII2CFC2 complex (phi F = 2.0 +/- 0.2 ns and phi S = 93 +/- 9 ns). Control experiments established that the RII2CFC2 complex was fully activated by cAMP at the same concentrations (0.2-0.4 microM) used for the anisotropy measurements. Together, the results demonstrate (1) that cAMP can induce the catalytic activity of cAPK without subunit dissociation and (2) that cAMP binding to holoenzyme is insufficient to explain its in vivo dissociation.

Catalysis↗
PubMed1995

Human mast cell tryptase isoforms: separation and examination of substrate-specificity differences.

Tryptases are trypsin-like enzymes found in mast cell granules that appear to exist as tetramers. These enzymes are not controlled by blood plasma proteinase inhibitors and only cleave a few physiological substrates in vitro, including high-molecular-mass kininogen (HMMK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) contained two bands of approx. molecular mass 29 and 33 kDa on SDS/PAGE. These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high-molecular-mass form (high-HLT) being eluted with 10 microM heparin and the low-molecular-mass form (low-HLT) subsequently eluted with 1 M NaCl. Removal of asparagine-linked carbohydrate caused both isoforms to run as single sharp bands on SDS/PAGE, differing slightly in molecular mass. Separation of these two isoforms of tryptase shows that tetramers consist of four homologous subunits rather than mixtures of the two isoforms. Using HMMK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMMK at Arg-431 within the C-terminal anionic binding region of the molecule, whereas low-HLT cleaved HMMK simultaneously at multiple sites within the C-terminal portion of the molecule. On the basis of HPLC peptide mapping, each isoform also cleaved VIP at different sites. Comparison of cleavage rates based on the active-site concentrations of titrated isoforms showed that low-HLT cleaved HMMK more rapidly than did high-HLT. These two isoforms may represent different gene products or they may result from post-translational modification.

Amino Acid Sequence↗
PubMed1995

Differential agonist-induced displacement of quinacrine and ethidium from their respective histrionicotoxin-sensitive binding sites on the Torpedo acetylcholine receptor.

Fluorescence spectroscopy was used to begin to localize the agonist inhibitory binding site on the nicotinic acetylcholine receptor (AcChR) from Torpedo californica. High concentrations of three cholinergic agonists, suberyldicholine (SubCh), acetylcholine (AcCh), and carbamylcholine (CCh), differentially inhibited the binding of two noncompetitive inhibitors (NCIs), quinacrine and ethidium, which bind at distinctly different loci on the desensitized AcChR at zero membrane potential. The agonist-induced inhibition of quinacrine binding occurred at significantly lower (17-fold) concentrations than the inhibition of ethidium binding. Schild plots of SubCh inhibition of ethidium and quinacrine binding showed the competitive nature of the agonist inhibition of the binding of these two NCIs. The quenching constants for short-range quenching of receptor-bound quinacrine and ethidium fluorescence by spin-labeled acetylcholine were about the same as their inhibition constants for agonist-induced displacement of AcChR-bound quinacrine and ethidium. The results demonstrate that agonists can directly bind to both the quinacrine and the ethidium binding sites, albeit at different agonist concentrations. Because the agonist-induced displacement of receptor-bound quinacrine occurs at significantly lower concentrations than the displacement of ethidium, the quinacrine binding site is more likely than the ethidium binding site to form part of the agonist inhibitory binding site.

Acetylcholine↗
PubMed1995

Soluble CD14 truncated at amino acid 152 binds lipopolysaccharide (LPS) and enables cellular response to LPS.

CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. In order to identify the domains in CD14 required for function, we deleted increasing amounts of CD14 from the C terminus. Truncated CD14 cDNA sequences were transfected into COS-7 cells and serum-free conditioned medium was analyzed for mutant CD14 expression and bioactivity. Mutant CD14s containing as few as 152 amino acids were found to have activity equivalent to full-length sCD14. To further characterize the mutant CD14, we constructed a stable Chinese hamster ovary cell line expressing sCD14(1-152) and purified the protein to homogeneity. sCD14(1-152) bound radioactive LPS, enabled U373 cells to synthesize interleukin 6 in response to LPS, and enabled human neutrophils to respond to smooth LPS. In all of these assays, the behavior of sCD14(1-152) was quantitatively similar to full-length sCD14. We also found that two neutralizing anti-CD14 antibodies (3C10 and MEM-18) bound and neutralized sCD14(1-152). We conclude from these experiments that the N-terminal 152 amino acids of CD14 are sufficient to bind LPS and confer essentially wild-type bioactivity in vitro.

Amino Acids↗
PubMed1995

The relationship of corneal epithelial defect size to drug penetration.

OBJECTIVE: To determine the relationship between corneal epithelial defect size and corneal penetration of a triazole antifungal drug in an animal model. METHODS: Corneas of adult rabbits were débrided of epithelium 25%, 50%, 75%, or 100% of surface area; the untreated fellow eye served as a control. Tritiated saperconazole was applied to each cornea every 5 minutes for 1 hour. The animals were killed and the cornea and aqueous of each eye were assayed for radiolabel activity. RESULTS: Removal of 25% of the corneal epithelium produced an increase in corneal saperconazole concentration compared with eyes with intact epithelium. Increasing epithelial defect size from 25% to 50% produced a ninefold increase in mean corneal drug concentration (P = .0001). There was no further increase in corneal drug levels in eyes with 75% or 100% epithelial defects. A similar threshold effect was observed in aqueous drug concentration between 25% and 50% débridement (P = .0001). CONCLUSION: In this experimental model, an apparent threshold was noted between 25% and 50% epithelial defect area, beyond which larger defects did not significantly increase drug penetration into the cornea or aqueous. This may be of clinical benefit in circumstances in which epithelial débridement is considered to enhance drug penetration.

Animals↗
PubMed1995

Reversal of scopolamine induced amnesia in rats by the steroid sulfatase inhibitor estrone-3-O-sulfamate.

The intent of the study was to determine whether altering the metabolism of neurosteroids via blockade of the enzyme, steroid sulfatase, could enhance retention test performance in rats. The steroid sulfatase inhibitor estrone-3-O-sulfamate (EMATE) was administered alone and in combination with the neurosteroid dehydroepiandrosterone sulfate (DHEAS) to rats which were then tested for the reversal of scopolamine induced amnesia. EMATE enhanced the reversal of amnesia by DHEAS as measured by a passive avoidance test. When administered without DHEAS, as a single acute dose, EMATE had no effect. When administered without DHEAS over 10 consecutive days, however, EMATE significantly improved retention. These results suggest that steroid sulfatase inhibition can potentiate the memory enhancing properties of DHEAS. The study also suggests that increasing the levels of endogenous sulfated neurosteroids via the inhibition of steroid sulfatase activity may enhance learning and/or memory function.

Amnesia↗
PubMed1995

P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin.

Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P-selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P-selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein-dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL-1 must interact with P-selectin in order for neutrophils to roll on P-selectin at physiological shear stresses.

Amino Acid Sequence↗
PubMed1995

Effects of protein deficiency and diet consistency on the parotid gland and parotid saliva of rats.

Protein deficiency results in an increased susceptibility to dental caries, suggesting that oral host-defense properties are compromised. An important component of oral host defense is salivary gland function, which is affected by both protein deficiency and diet consistency. This study describes the effects on rat parotid gland growth and secretory function induced by feeding rats diets of normal (20%) or moderately low (7%) protein content, provided in either a powdered or solid form. In addition, since protein deficiency may result in a secondary zinc deficiency which, in turn, may affect salivary gland function, the effects of these diets on liver zinc concentration were also measured. From 22 to 47 days of age, rats (18/group) were fed the following diets: normal protein, powdered; normal protein, solid; low protein, powdered; and low protein, solid. With each diet consistency, liver zinc was higher for the normal protein group. Within each protein level, liver zinc was higher for the solid diet. This latter observation suggests that food mastication and the resultant stimulation of salivary gland function may also play a role in zinc metabolism. With the normal-protein diet, parotid gland weight was higher for the solid diet; with the low-protein diet, parotid gland weight was similar for both consistencies and did not differ from that of the group fed the "normal protein, powdered" diet. For both consistencies, parotid saliva protein concentration was greater for malnourished rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Reduction of toxicity of a vinca alkaloid by an anti-vinca alkaloid antibody.

Inadvertent oncolytic overdoses occur rarely, but can have serious consequences. We have investigated the possibility of using an antibody, 27.8.1A, reactive with vinca alkaloids, as a means of reducing the toxicity associated with overdose situations. In vitro cytotoxicity of a vinca derivative, 4-desacetyl- vinblastine-3-carbox-hydrazide (DAVLBHYD), with and without the addition of 27.8.1A, was determined. Using CCRF-CEM, a human acute lymphoblastic leukemia cell line, as a target in this assay, we observed a greater than 90% increase in cell viability using 100 micrograms/ml 27.8.1A with a 0.1 microgram/ml concentration of DAVLBHYD. 27.8.1A had no effect on cell viability when doxorubicin was used as a control drug in this assay. Similarly, the addition of an irrelevant antibody. EGFrL11, had no effect on the toxicity of DAVLBHYD. In an in vivo survival experiment, nude mice were injected with a toxic dose of DAVLBHYD and subsequently given four doses of 27.8.1A. All anti vinca antibody-treated mice survived, in contrast to the untreated group or irrelevant antibody-treated group in which only 25% and 10% of the mice survived, respectively.

Animals↗
PubMed1994

The histrionicotoxin-sensitive ethidium binding site is located outside of the transmembrane domain of the nicotinic acetylcholine receptor: a fluorescence study.

A novel, relatively photostable, long-wavelength fluorescent membrane probe, N-(Texas Red sulfonyl)-5(and 6)-dodecanoylamine (C12-Texas Red), was synthesized and used as an electronic energy acceptor for Förster fluorescence resonance energy transfer (FRET) between ethidium bound to a histrionicotoxin-sensitive binding site on the Torpedo nicotinic acetylcholine receptor (AChR) and the lipid membrane surface. FRET from membrane-partitioned 5-(N-dodecanoylamino)fluorescein (C12-fluorescein) to the membrane-partitioned C12-Texas Red was also determined with a parallel set of cuvettes to (1) compare FRET results with a donor in a known position in the membrane and (2) assess the surface density of the membrane-partitioned C12-Texas Red. Stern-Volmer analysis of the FRET results showed that C12-Texas Red quenched membrane-partitioned C12-fluorescein fluorescence 2.9 times more effectively than it quenched the receptor-bound ethidium fluorescence even though the Förster critical distances for the two donor-acceptor pairs were very similar (49.9 and 54.3 A, respectively). Analysis of the ethidium to C12-Texas Red FRET as a function of acceptor surface density with the assumptions that the donor is attached along the major axis of symmetry of a cylindrical protein embedded perpendicularly into the membrane (On-Axis FRET model) suggested that the distance of closest approach between the receptor-bound ethidium and the membrane surface was approximately 52 A. Because the minimum distance between the surface of the lipid-membrane domain and the major symmetry axis of the AChR is approximately 28 A, the FRET results strongly suggest that the ethidium binding site is not located near the entrance of the luminal transmembrane domain is generally assumed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphibian Venoms↗
PubMed1994

Afamin is a new member of the albumin, alpha-fetoprotein, and vitamin D-binding protein gene family.

A novel human serum protein with a molecular mass of 87,000 daltons was purified to homogeneity and subjected to amino acid sequence analyses. These sequences were used to design oligonucleotide primers and to isolate a full-length cDNA. The amino acid sequence encoded by the cDNA shares strong similarity to albumin family members and shares the characteristic pattern of Cys residues observed in this family. In addition, the gene maps to chromosome 4 as do other members of the albumin gene family. Based upon these observations, we conclude that the 87,000-dalton protein, which we designate afamin (AFM), is the fourth member of the albumin family of proteins. Afamin cDNA was stably transfected into Chinese hamster ovary cells and recombinant protein (rAFM) was purified from conditioned medium. Both rAFM and AFM purified from human serum react with a polyclonal antibody that was raised against a synthetic peptide derived from the deduced amino acid sequence of AFM.

Albumins↗
PubMed1994

Antibody-induced conformational changes in the Torpedo nicotinic acetylcholine receptor: a fluorescence study.

Quantitative fluorescence spectroscopy was used to develop a structural picture of the effects of two monoclonal antibodies (mAbs) on the conformation of the Torpedo nicotinic acetylcholine receptor (nAChR). The two mAbs (A6 and B1) examined selectively blocked ligand binding to either the high-affinity (A) or the low-affinity (B) binding sites for agonists/competitive antagonists. The distances between dansyl-C6-choline bound to the unblocked agonist/competitive antagonist binding site and one of two lipophilic probes (C12-eosin or C18-rhodamine) partitioned into the lipid membrane were estimated by using fluorescence resonance energy transfer. Control experiments demonstrated that both mAbs decreased the affinity and fluorescence lifetime of receptor-bound dansyl-C6-choline. The binding of the B1 mAb to the B site did not significantly change the calculated distance between the unblocked A binding site and the membrane surface. However, the binding of the A6 mAb to the A site induced the B site to move into close proximity to the lipid membrane. This conformational change was confirmed by a 45-fold increase in the paramagnetic quenching of the B-site-bound dansyl-C6-choline fluorescence by lipid-intercalated 5-doxylstearate. The results indicate that these mAbs not only selectively block ligand binding to either the A or the B acetylcholine sites but also, in the case of the A6 mAb, induce global conformational changes of the receptor, which appear to involve a movement of the B binding site into close proximity of the lipid membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Human mast cell tryptase activates single-chain urinary-type plasminogen activator (pro-urokinase).

Human lung mast cell tryptase is a trypsin-like serine proteinase that is stored in mast cell granules and released by activated mast cells. Here we report that mast cell tryptase is a potent activator of single-chain urinary-type plasminogen activator (scu-PA, or prourokinase), the zymogen form of urinary-type plasminogen activator (u-PA). Activation was complete within 75 min using an enzyme:substrate molar ratio of 1:50 and was accompanied by cleavage of scu-PA at Lys158-Ile159, generating active two-chain u-PA. The reaction was dependent on enzyme concentration and obeyed Michaelis-Menten kinetics. Kinetic constants calculated for scu-PA activation by mast cell tryptase are Km = 34 microM, Vmax = 3.6 pmol of u-PA/min, and kcat = 0.08 s-1. These data suggest that tryptase from tumor-associated mast cells may participate in the activation of scu-PA.

Amino Acid Sequence↗
PubMed1994

Total synthesis and biological properties of novel antineoplastic (chloromethyl)furanoindolines: an asymmetric hydroboration mediated synthesis of the alkylation subunits.

1,2-Dihydro-1-(chloromethyl)-5-hydroxy-8-methyl-3H-furano[3,2-e]in dole (CFI) as a novel replacement of the cyclopropylpyrroloindoline (CPI) alkylation subunit of CC-1065, U-71184, and U-73975 (adozelesin) has been synthesized and incorporated into a series of efficacious antineoplastic agents. A partial solution to an asymmetric synthesis of the CFI alkylation subunit has been achieved by the implementation of an asymmetric hydroboration reaction of an intermediate 3-methyleneindoline (13). Extension to the asymmetric synthesis of the CBI and CI alkylation subunits is presented. The demonstration and comparative study of the sequence-selective DNA alkylation properties of the CFI-based agents are detailed, and the preliminary in vitro and in vivo antineoplastic properties of these agents in the human epidermoid cell lung carcinoma (T222) are described.

Alkylation↗
PubMed1994

Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14.

CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.

Acute-Phase Proteins↗
PubMed1994

Basal acid output and gastric acid hypersecretion in gastroesophageal reflux disease. Correlation with ranitidine therapy.

The purpose of this study was to evaluate possible differences in basal gastric acid secretion with regard to severity of gastroesophageal reflux disease. Basal acid output was determined by nasogastric suction in 228 patients with gastroesophageal reflux disease who received upper gastrointestinal endoscopy and were diagnosed with either pyrosis alone (N = 98), erosive esophagitis with or without pyrosis (N = 87), or Barrett's esophagus (N = 43). Mean basal acid output for the 228 patients with gastroesophageal reflux disease was 6.5 +/- 5.6 meq/hr, which was significantly different from 65 normal subjects with a mean basal acid output of 3.0 +/- 2.7 meq/hr (P < 0.0001). Compared to normal subjects, mean basal acid outputs significantly differed for patients with pyrosis (P < 0.05), esophagitis (P < 0.01), and Barrett's esophagus (P < 0.01). There was also a significant difference in mean basal acid output between the patients with pyrosis and Barrett's esophagus (P < 0.01). Nineteen of the 98 patients with pyrosis (19%), 24 of the 87 patients with esophagitis (28%), and 15 of the 43 patients with Barrett's esophagus (35%) had gastric acid hypersecretion (basal acid output greater than 10.0 meq/hr). One hundred forty-six patients with gastroesophageal reflux disease were treated with ranitidine in doses that resulted in complete healing of esophagitis and disappearance of pyrosis. Ninety-three patients responded to ranitidine 300 mg/day; however, 53 patients required increased dose of ranitidine (mean 1205 mg/day, range 600-3000 mg/day).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Transverse distance between the membrane and the agonist binding sites on the Torpedo acetylcholine receptor: a fluorescence study.

Fluorescence dipolar resonance energy transfer between a receptor-bound fluorescent agonist, dansyl-C6-choline, and two membrane-partitioned fluorescent probes, C18-rhodamine and C12-eosin, was used to measure the transverse distance between the acetylcholine (ACh) binding sites on the intact Torpedo nicotinic acetylcholine receptor (nAChR) and the surface of the lipid membrane. Control experiments demonstrated that: (1) dansyl-C6-choline binds to cobra-alpha-toxin sensitive sites on the nAChR with a KD approximately 20 nM, (2) the quantum yield of dansyl-C6-choline increases 3.1-fold upon binding, and (3) the receptor-bound dansyl-C6-choline fluorescence is stable for at least 2 h. The calculated transverse distances between receptor-bound dansyl-C6-choline and the membrane-partitioned acceptors, C12-eosin and C18-rhodamine, were 31 and 39 A, respectively. Therefore, given the dimensions of the extracellular domain of the receptor, the ACh binding sites are located significantly below (approximately 25 A) the extracellular apex of the nAChR. These results are in agreement with the recent proposed location for the ACh binding sites in a pocket within each of the two alpha-subunits, approximately 30 A above the membrane surface (Unwin, N. (1993) J. Mol. Biol. 229: 1101-1124).

Animals↗