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Biomedical subjects

D A Johnson

Publications and source records attributed to D A Johnson.

At least 199 records · Page 11Linked to original sources
PubMed1990

Organization of the 5S rRNA genes in the soybean Glycine max (L.) Merrill and conservation of the 5S rDNA repeat structure in higher plants.

The 5S rRNA gene of the soybean Glycine max (L.) Merr. has been cloned on a 556-bp fragment of DNA and sequenced. This fragment contains two copies of the soybean 5S rDNA sequence, one intact and one truncated, separated by noncoding DNA. We have used this clone to investigate the organization of the 5S genes within the soybean genome and the extent of their methylation. Our results demonstrate that soybean 5S genes are clustered, organized into tandem repeats of 330 bp, and extensively methylated. Hybridization of the 5S sequence to Southern transfers of soybean DNA digested with BamHI reveals a striking ladderlike pattern. Hybridization of the soybean 5S sequence to a wide variety of plant DNAs results in similar patterns, suggesting that the 5S rDNA sequence, gene organization, and methylation pattern are conserved in many higher plants.

Base Sequence↗
PubMed1990

Inhalation of nitrogen dioxide fails to reduce the activity of human lung alpha-1-proteinase inhibitor.

Healthy, nonsmoking human volunteers were exposed to environmentally relevant concentrations of NO2 followed by bronchoalveolar lavage (BAL) to study whether NO2 exposure decreases the functional activity of alpha-1-proteinase inhibitor (alpha 1-PI) in the lung. Two 3-h exposure protocols with intermittent exercise were employed and BAL was performed 3.5 h after exposure. The first exposure protocol with nine subjects involved three 2-ppm "peaks" with a 0.05 ppm background, whereas the second protocol with 15 subjects was a continuous exposure to 1.5 ppm NO2. All subjects were randomly exposed to either air or NO2, with at least a 2-wk interval between treatments, and the BAL fluids obtained after air exposure served as the controls. The BAL fluids were analyzed for alpha 1-PI elastase inhibitory activity, the immunologic concentration of alpha 1-PI, total protein, and albumin. The ratio of alpha 1-PI activity to its immunologic concentration was taken as the functional activity of alpha 1-PI, and possible changes in the amount of alpha 1-PI in the lung were assessed by examining the ratio of the immunologic concentration of alpha 1-PI to total protein. Neither of the NO2 exposure protocols resulted in a decrease in the functional activity of alpha 1-PI, nor were there alterations in the immunologic levels of alpha 1-PI. These data suggest that short-term exposures to low levels of NO2 do not result in a lung-localized deficiency of active alpha 1-PI, which has been hypothesized to be a contributing factor in the pathogenesis of emphysema.

Administration, Inhalation↗
PubMed1990

Drug prescribing for schizophrenic out-patients on depot injections. Repeat surveys over 18 years.

Serial surveys of the prescribing practices of psychiatrists for schizophrenic out-patients over 1970-88 showed important changes. During the interval 1970-83, polypharmacy and the use of multiple neuroleptics were reduced. The total dose of neuroleptics prescribed fell and the proportion administered by depot injection increased. Since 1983, however, some of these favourable trends have been reversed. Prescribing practices may also vary between teaching and non-teaching hospitals.

Adolescent↗
PubMed1990

Oxidant effects on rat and human lung proteinase inhibitors.

This project tested the hypothesis that inhaled oxidants could cause lung damage by inactivating the proteinase inhibitors that normally protect the lung from proteolysis. Rat alpha-1-proteinase inhibitor (alpha 1-PI)2 was purified from blood plasma, and antibodies to this inhibitor were prepared. The activity of alpha 1-PI in lung lavage fluids from rats was measured by elastase inhibition, and the immunological concentration of alpha 1-PI was quantified in an enzyme-linked immunoassay. The ratio of the amount of active alpha 1-PI relative to its immunological concentration was examined as a measure of the inhibitor's functional activity. This ratio and the ratio of the immunological concentration of alpha 1-PI to the total protein concentration were determined in lung lavage fluids from rats exposed to air, 10 parts per million (ppm) nitrogen dioxide, and diesel emissions (3.5 mg/m3 particulates) for 12, 18, and 24 months. Only diesel exposures resulted in a statistically significant reduction in the functional activity of alpha 1-PI of 30 percent (p less than 0.05). Similar studies were performed on rats exposed to nitrogen dioxide (0.5 ppm background with peaks of 1.5 ppm) and ozone (0.06 ppm background with peaks of 0.25 ppm) for 12 and 18 months. No statistically significant effects were observed in the functional activity of alpha 1-PI or its immunological concentration. In other protocols, rats were acutely exposed to 0.8 ppm or 1.2 ppm ozone for two, four, or eight hours, and to 0.5 ppm or 0.8 ppm ozone in conjunction with 8 percent carbon dioxide for two or seven hours. Although these acute exposure conditions did not reduce the functional activity of alpha 1-PI, the immunological concentration of alpha 1-PI and the elastase inhibitory activity, relative to other proteins, were significantly increased in relation to the total amount of ozone inhaled. The functional activity of alpha 1-PI also was measured in the bronchoalveolar lavage fluids of human subjects exposed to nitrogen dioxide (0.05 ppm with 2 ppm peaks, or to 1.5 ppm continuously) for three hours and to ozone (0.4 ppm) for two hours during exercise. These exposures did not result in significant changes in the functional activity of alpha 1-PI or its immunological concentration.(ABSTRACT TRUNCATED AT 400 WORDS)

Air Pollutants↗
PubMed1990

A prospective study of the prevalence of colonic neoplasms in asymptomatic patients with an age-related risk.

This study was designed to determine the prevalence of colorectal neoplasia in healthy, asymptomatic adults with an age-related risk for colorectal neoplasia. Ninety patients were studied with air contrast barium enema and colonoscopy. The study population included 61 males and 21 females, with an age range of 51-82 yr (65 +/- 2 yr). Adenomatous polyps were found in 27% of males and 14% of females or 24% of patients overall. Sixty-six percent of these neoplasms were above the rectosigmoid junction and the mean size of the polyps was 6.5 +/- 1.2 mm. In two patients, carcinoma was discovered. A linear association between age and the prevalence of colonic neoplasia was not demonstrated. This study demonstrates a relatively high prevalence of colonic neoplasia in patients with an age-related risk.

Adenoma↗
PubMed1990

Sincalide-augmented quantitative hepatobiliary scintigraphy (QHBS): definition of normal parameters and preliminary relationship between QHBS and sphincter of Oddi (SO) manometry in patients suspected of having SO dysfunction.

Sphincter of Oddi (SO) dysfunction presents with vague abdominal pain and/or abnormal liver function tests, and is presumably due to SO stenosis or spasm. Clinical, laboratory, and imaging methods of diagnosis have been less than ideal. Initially, we determined normal quantitative hepatobiliary scintigraphy (QHBS) parameters both pre- and post-sincalide administration. Thirty-one "normals" were analyzed, and post-sincalide common bile duct (CBD) dynamics could be satisfactorily determined in 29 (94%) subjects. Normal values at sincalide-augmented QHBS are reported. Next, 10 patients suspected of having SO dysfunction were studied prospectively using SO manometry and QHBS. The two tests were in agreement in seven cases (4: normal CBD dynamics, 3: abnormal). In one case of advanced SO stenosis, QHBS was abnormal, but SO manometry could not be performed. In the two remaining cases, SO manometry and QHBS gave discordant results. Of greatest importance, no significant correlation existed between the quantitative parameters of these two tests. Sincalide-augmented QHBS is possible and may, in the future, be of value in the diagnosis of SO dysfunction and/or partial CBD obstruction.

Adult↗
PubMed1989

Fragments of bovine insulin-like growth factors I and II stimulate proliferation of rat L6 myoblast cells.

The active sites of bovine insulin-like growth factor (IGF) I and II fragments were studied. Overlapping fragments of IGF I (residues 1-25, 11-35, 21-45, 31-55, and 41-70) and of IGF II (residues 1-24, 10-34, 20-44, 30-54, and 40-67) were chemically synthesized. The activity of the fragments was measured by stimulating the proliferation of rat L6 myoblast cells. Two fragments of IGF I (residues 21-45 and 31-55) and two fragments of IGF II (residues 20-44 and 30-54) were active while the other fragments were inactive in stimulating cell proliferation. Although the activity of these fragments was observed only at a high concentration of 0.1 mM, the results imply that the active site is located around residues 31-45 for IGF I fragments and residues 30-44 for IGF II fragments. Consequently, an IGF I fragment (residues 26-50) having a five-residue extension to both the N- and C-terminal sites of residues 31-45 also stimulated the proliferation of L6 myoblast cells. Furthermore, the substitution of Ile-35 in two IGF II fragments (residues 21-45 and 31-55) by Ser inactivated these fragments. This suggests that Ile-35 is an essential residue for IGF II fragment activity. Ser-35, which was reported in the original sequencing of bovine IGF II, is incorrect in the sequence and furthermore has been consistently found to be an Ile-35 in our hands.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Plastocyanin cytochrome f interaction.

Spinach plastocyanin and turnip cytochrome f have been covalently linked by using a water-soluble carbodiimide to yield an adduct of the two proteins. The redox potential of cytochrome f in the adduct was shifted by -20 mV relative to that of free cytochrome f, while the redox potential of plastocyanin in the adduct was the same as that of free plastocyanin. Solvent perturbation studies showed the degree of heme exposure in the adduct to be less than in free cytochrome f, indicating that plastocyanin was linked in such a way as to bury the exposed heme edge. Small changes were also observed when the resonance Raman spectrum of the adduct was compared to that of free cytochrome f. The adduct was incapable of interacting with or donating electrons to photosystem I. Peptide mapping and sequencing studies revealed two sites of linkage between the two proteins. In one site of linkage, Asp-44 of plastocyanin is covalently linked to Lys-187 of cytochrome f. This represents the first identification of a group on cytochrome f that is involved in the interaction with plastocyanin. The other site of linkage involves Glu-59 and/or Glu-60 of plastocyanin to as yet unidentified amino groups on cytochrome f. Euglena cytochrome c-552 could also be covalently linked to turnip cytochrome f, although with a lower efficiency than spinach plastocyanin. In contrast, a variety of cyanobacterial cytochrome c-553's and a cyanobacterial plastocyanin could not be covalently linked to turnip cytochrome f.

Amino Acid Sequence↗
PubMed1989

Distance between the agonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor possesses an agonist binding site on each of the two alpha-subunits and an allosterically coupled noncompetitive inhibitor (NCI) site. The spatial relationships between these sites have been determined by fluorescence energy transfer employing lifetime and steady-state techniques with two donor-acceptor pairs. 6-(5-Dimethylaminonaphthalene-1-sulfonamido)hexanoic acid-beta-(N-trimethylammonium)ethyl ester (dansyl-C6-choline, an agonist) and bis(choline)-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)-iminodiprop rionate (BCNI, a competitive antagonist) were employed as energy donors bound to the agonist sites. Ethidium was employed as a specific probe of the NCI site and served as the energy acceptor for both donors. Under steady-state conditions, energy transfer was measured by monitoring BCNI fluorescence as a function of occupancy of ethidium. Changes in acceptor occupancy were achieved by titrating acetylcholine receptor-donor-acceptor complexes with phencyclidine, a nonfluorescent NCI ligand. Extrapolation of the data to 100% acceptor occupancy yielded a transfer efficiency of 38% for the BCNI-ethidium pair. In the second method, the transfer efficiency of the dansyl-C6-choline-ethidium pair was determined by analysis of the reduction of the donor-excited state fluorescence lifetime. The nanosecond decay rates for dansyl-C6-choline measured in the presence of phencyclidine are characterized by two lifetimes (tau 1 = 6.7; tau 2 = 17.1 ns) with an amplitude ratio, alpha 1/alpha 2 = 2.3. In the presence of ethidium, the two lifetimes were proportionally diminished while retaining a comparable ratio of amplitudes. Displacement of ethidium from the NCI site by phencyclidine restored the two lifetimes to their original values. These data indicate that the donors bound to the two agonist sites transferred energy with similar efficiencies to the acceptor. Thus, the lifetime data suggest that the NCI site is approximately equidistant from each of the agonist sites. The corrected efficiency of donor quenching by this method was 34%, a value in close accord with the steady-state measurements. The distance between the agonist sites and the NCI site was calculated to be between 21-35 A for the BCNI/ethidium pair and 22-40 A for the dansyl-C6-choline/ethidium pair. Consideration of these distances with respect to the molecular dimensions of the receptor and location of the agonist sites suggests a location for the NCI site near the ion channel at the extracellular surface of the membrane bilayer.

Animals↗
PubMed1989

Fluorescence energy transfer between cysteine 199 and cysteine 343: evidence for MgATP-dependent conformational change in the catalytic subunit of cAMP-dependent protein kinase.

The catalytic subunit of cAMP-dependent protein kinase has two cysteine residues, Cys 199 and Cys 343, which are protected against alkylation by MgATP [Nelson, N. C., & Taylor, S. S. (1981) J. Biol. Chem. 256, 3743]. While Cys 199 is in close proximity to the active site of the catalytic subunit and is probably directly protected against alkylation by MgATP, the mechanism by which MgATP prevents alkylation of Cys 343 is unclear. To determine whether MgATP directly protects Cys 343 from alkylation by being in close proximity to both Cys 199 and the MgATP binding site, fluorescence resonance energy transfer techniques were used to measure the distance between Cys 199 and Cys 343. Two different donor-acceptor pairs containing 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole at Cys 199 as the acceptor and either 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2, 8- dione or N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine at Cys 343 as the donor were prepared following the method described in the preceding paper [First, E. A., & Taylor, S. S. (1989) Biochemistry (preceding paper in this issue)]. From the efficiencies of fluorescence resonance energy transfer for each donor-acceptor pair, the distance between Cys 199 and Cys 343 was estimated to be between 31 and 52 A. Since Cys 199 is close to the MgATP binding site and since MgATP cannot extend beyond a distance of 16 A, it is unlikely that Cys 343 at a distance of at least 31 A from Cys 199 is in direct contact with the bound nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1989

Sodium dodecyl sulfate enhancement of quantitative immunoenzyme dot-blot assays on nitrocellulose.

Treating proteins with low concentrations of sodium dodecyl sulfate (SDS) and boiling for 2-3 min increased the linear range and total amount of protein that could be bound to nitrocellulose. Human serum albumin (HSA) and cathepsin G (Cat G) were both optimally bound at an SDS concentration of 10 micrograms/ml, while bronchial leukocyte proteinase inhibitor (BLPI) required 50 micrograms/ml SDS for optimum binding, corresponding to SDS-to-protein weight ratios of 0.5 and 2.5, respectively. Ionic strength and pH of the blotting buffers had a greater effect on the binding of SDS-treated proteins than on native proteins, with the linear binding range and total capacity for SDS-treated proteins being increased. Boiling SDS-treated human leukocyte extracts inactivated endogenous peroxidases, eliminating their interference with peroxidase-linked secondary antibodies in immunoassays. The nonionic detergents, Tween 20 and Nonidet P-40, were shown to rapidly wash both native and SDS-treated HSA off the filters, but these HSA samples were stable to washing with SDS. Although SDS-treated Cat G was more stable with nonionic detergents than was native Cat G, it was less resistant to washing with SDS. The substitution of SDS for nonionic detergents improved the response of immunoassays with native and SDS-treated proteins. Affinity-purified antibodies to human mast cell tryptase cross-reacted with native Cat G, but not with SDS-treated Cat G, indicating that SDS treatment can improve the specificity of immunoassays employing polyclonal antisera. These effects appear to be the result of partial denaturation and increases in the hydrophobicity of SDS-treated relative to native proteins.

Buffers↗
PubMed1989

Effects of dietary protein on food and water intake in spontaneously hypertensive rats.

We have been studying the development of hypertension in spontaneously hypertensive rats (SHR) fed a low protein diet. The effects of a low protein diet upon food and water intake were examined. Body weight gain, food and water intake were measured in three to twenty-three week-old SHR and Wistar Kyoto rats (WKY) fed diets containing 8%, 15% or 25% casein. Body weights of SHR and WKY fed an 8% casein diet were significantly lower at 23 weeks than rats on the higher protein diets, although both groups on the 8% diet consumed more food and water per g of body weight. In addition, SHR fed an 8% casein diet drank less water per gram of food than WKY or SHR fed 15% and 25% casein diets. These results indicate that changes in food and water intake, as a consequence of low protein diets, should be an additional consideration when examining the effects of dietary protein on the development of hypertension.

Animals↗
PubMed1989

5-bromodeoxyuridine (BUdR) quenching of acridine orange fluorescence distinguishes cycling and non-cycling normal and malignant bone marrow cells in vitro.

Suppression of the green fluorescence of acridine orange by 5-bromodeoxyuridine incorporation into cellular DNA was measured by flow cytometry. Bone marrow cells from normal volunteers and patients with chronic myelogenous leukemia acute lymphocytic leukemia acute myelogenous leukemia and multiple myeloma were incubated with BUdR in vitro. By 24 h acridine orange stained cycling cells that had synthesized DNA in the presence of BUdR were differentiated from quiescent cells as a second peak with quenched green fluorescence (DNA). After 72 h in culture 11-65% of the G0/G1 cells from normal bone marrows and bone marrows with myeloid leukemia were identified as cycling in culture by the presence of a second peak with quenched green fluorescence. A greater percentage of cells with BUdR quenched AO fluorescence was associated of acridine orange was higher in the cycling cells that had synthesized DNA in the presence of BUdR than in the non-cycling G0/G1 cells. In one patient with AML there was quenching of the DNA fluorescence of the aneuploid population but not the diploid population indicating that the aneuploid leukemia cells were proliferating. In contrast in patients with multiple myeloma, the quenched fluorescence of the diploid cell population and not the aneuploid cells, indicated that the diploid cells were proliferating. The cells from patients with untreated ALL failed to proliferation prohibiting an in vivo assessment of growth. Although measurements of proliferation obtained by this method are clearly influenced by the cell's adaptation to culture, measurement of BUdR quenching of acridine orange fluorescence is technically feasible and can identify and allow characterization of the cycling population of cells.

Acridine Orange↗
PubMed1989

New antitumor monoclonal antibody-vinca conjugates LY203725 and related compounds: design, preparation, and representative in vivo activity.

A method has been developed to allow the direct coupling of the cytotoxic vinca alkaloid 4-desacetylvinblastine-3-carbohydrazide (DAVLB hydrazide) to a variety of murine monoclonal antibodies directed against human solid tumors. Periodate oxidation of carbohydrate residues on the antibodies, followed by reaction with DAVLB hydrazide in aqueous acid affords, in most cases, conjugates with conjugation ratios of 4-6 vincas per antibody in high yield without significantly impairing antigen binding or solubility. The outcome of the conjugation reaction is highly dependent on the concentration of, and time of exposure of the protein to, the oxidant. These conjugates exhibit potent antitumor activity in vivo against a number of human solid tumor-nude mouse xenografts, with efficacy and safety increased over unconjugated DAVLB hydrazide. This antitumor activity is also superior to that of similarly prepared but nontarget tumor binding antibody-DAVLB hydrazide conjugates. MoAb-DAVLB hydrazide conjugates release DAVLB hydrazide in solution in a temperature- and pH-dependent manner. Hydrolytic release of unmodified DAVLB hydrazide from tumor-localized MoAb-DAVLB hydrazide conjugates in vivo may be an important factor in their antitumor activity.

Animals↗
PubMed1989

Fecal occult blood testing. Problems, pitfalls, and diagnostic concerns.

The diagnostic approach to the asymptomatic patient who has tested positive for fecal occult blood varies among clinicians. Colonoscopic evaluation is the procedure of choice in initiating the workup of patients 50 years of age and older. In younger patients, examination by sigmoidoscopy and barium enema using air for contrast (double contrast study) may be considered as an alternative to colonoscopy as the initial test. Evaluation of the upper gastrointestinal tract should be considered if a colonic source is not detected. The challenge in diagnostic test selection lies in choosing the most appropriate and cost-effective procedure for detecting disease, especially neoplastic lesions.

Barium Sulfate↗
PubMed1989

Monoclonal antibody L/1C2 reactive with a human carcinoma associated antigen.

A murine IgG3 monoclonal antibody which defines a human carcinoma associated antigen is described. The antibody, designated L/1C2, was made against a human lung squamous cell carcinoma line designated USCLS-1. It reacts with the surface of 15 of 16 viable human carcinoma cell lines and was detected in 70 of 78 frozen sections of human carcinomas. Melanoma cell lines and frozen sections of melanomas and a lymphoma were unreactive. Normal tissue reactivity included vessels, plus some ducts, glandular structures, and epithelial surfaces. Similar normal tissue reactivity patterns were seen with Rhesus monkey tissue samples. Immunoprecipitation studies indicate that L/1C2 reacts with a glycoprotein doublet which migrates in the range of 110,000 to 140,000 Mr under reducing conditions. Fluorescence analysis suggests this antigen is internalized following reaction with the L/1C2 antibody. Using in vitro human tumor cell growth inhibition assays, it was possible to achieve significant growth inhibition with L/1C2, while another target cell-reactive antibody in the same assay had no inhibitory effect.

Adenocarcinoma↗
PubMed1989

Effect of diazoxide and hydralazine on intestinal transit and blood pressure in the rat.

This study examines the effects of diazoxide and hydralazine on gastrointestinal and vascular smooth muscle. The effect of the drugs administered individually and in combination was determined on small intestinal transit and blood pressure in adult male Sprague-Dawley rats. Both diazoxide and hydralazine produced dose-dependent decreases in the ratio of intestinal transit to total length. Additionally, combinations of various doses of hydralazine and diazoxide produced a greater than simple summation of the inhibitory effects on intestinal transit. The combination of diazoxide and hydralazine produced only a simple additive effect on mean arterial pressure.

Animals↗