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Biomedical subjects

D D Johnson

Publications and source records attributed to D D Johnson.

157 records · Page 9Linked to original sources
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Immunoassay reagents for thyroid testing. 1. Synthesis of thyroxine conjugates.

Immunoreagents were designed to improve the performance of a commercial fluorescent polarization immunoassay for thyroxine. The thyroxine immunogen was prepared by selective coupling of N-acetyl-L-thyroxine to BSA via an aminocaproic acid spacer arm. The fluorescent tracer was prepared by a multistep reaction sequence which relied on extensive use of orthogonal protecting groups.

Chromatography, High Pressure Liquid↗
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Characterization of protein-hapten conjugates. 1. Matrix-assisted laser desorption ionization mass spectrometry of immuno BSA-hapten conjugates and comparison with other characterization methods.

Several different low molecular weight haptens were conjugated to BSA to produce immunogens useful for antibody development. The extent of BSA modification due to covalent attachment of hapten was estimated by matrix-assisted laser desorption ionization mass spectrometry. The average number of hapten incorporated to immunogen was determined from the difference in the measured molecular weights of the conjugate from nonmodified BSA. The results from mass spectrometry were compared with results obtained from other more traditional methods of immunogen characterization (UV analysis, trinitrobenzenesulfonic acid titrations, and gel electrophoresis). In each case we were able to calculate the average number of hapten covalently bound to BSA for each synthetically prepared immunogen using matrix-assisted laser desorption ionization mass spectrometry. The other methods presented limitations in certain cases.

Electrophoresis, Polyacrylamide Gel↗
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Immunoassay reagents for thyroid testing. 3. Determination of the solution binding affinities of a T4 monoclonal antibody Fab fragment for a library of thyroxine analogs using surface plasmon resonance.

A library of thyroxine analogs and tracers was prepared, and their solution binding affinities for an anti-T4 Fab fragment were determined using a single high-density L-T4 biosensor surface in a BIAcore surface plasmon resonance instrument. The high-density L-T4 analog biosensor was calibrated by determination of the initial binding rate was of known concentrations of free anti-T4 Fab fragment in solution to the biosensor surface. A range of individual thyroxine analog and tracer concentrations was subsequently mixed with a fixed concentration of anti-T4 Fab fragment. The concentration of free anti-T4 Fab fragment in each solution at equilibrium was determined, and the equilibrium dissociation constant (KD) for each case was derived. The KD values determined in solution are compared to values determined by a direct kinetic analysis on the BIAcore instrument using individual biosensor surfaces.

Acridines↗
PubMedDate not supplied

Synthesis of 6beta-[(2'-Aminoethyl)carboxamidomethyl]estradiol and preparation of estradiol probes.

Reformatsky reaction of 3, 17beta-bis[(2-trimethylsilyl)ethoxymethyl]-1,3, 5(10)-estratrien-6-one (2) with bromoethyl acetate and zinc gave the ester (3) in 60% yield which upon treatment with methanesulfonyl chloride in pyridine afforded the olefinic esters (4 and 5) as an endo and exo mixture (67:33 ratio) in 81% yield. Hydrolysis of the SEM protective groups in compounds 4 and 5 followed by hydrogenation of the resulting hydroxy compounds 6 and 7 using 10% Pd/C afforded an epimeric mixture (beta:alpha = 79:21) of 6-[(ethoxycarbonyl)methyl]estradiol (8a and 8b) in 95% yield. Hydrolysis of the ethyl esters (8a and 8b) using sodium hydroxide gave the acid (9a and 9b) in 81% yield. The epimeric mixture of acids (9a and 9b) was activated, treated with tert-butyl-N-(2-aminoethyl)carbamate (10), and purified by HPLC to afford 6beta-[[[(2-tert-butoxycarbonyl)amino]ethyl]carboxamidomethyl] estradiol (11) in 39% yield as the major isomer. Hydrolysis of the BOC group in compound 11 using TFA afforded the desired 6beta-[(2-aminoethyl)carboxamidomethyl]estradiol 12 in 50% yield. The biotinylated estradiol probe 14, fluorescent probe 16, and chemiluminescent probe 18 were prepared from 6beta-[(2'-aminoethyl)carboxamidomethyl]estradiol (12) and the corresponding biotin, 5-carboxyfluorescein, and 10-(3-sulfopropyl)-N-tosyl-N-(3-carboxypropyl)acridinium-9-carboxamide N-succinimidyl esters (13, 15, and 17) in 65-74% yield and 99% purity.

Biotinylation↗
PubMedDate not supplied

Collagen cross-links. Synthesis of immunoreagents for development of assays for deoxypyridinoline, a marker for diagnosis of osteoporosis.

(+)-Deoxypyridinoline (Dpd, 2) is a cross-link of bone collagen, which is released and excreted in urine during process of bone resorption. It has been shown that this bone collagen degradation product, Dpd (2) is a useful marker for diagnosis of osteoporosis and other metabolic bone diseases. In this paper, the design and synthesis of two immunogens (3, 4) via of conjugation of succinimidyl ester (14) to carrier protein, bovine serum albumin, or keyhole limphet hemocyanin, was presented. Additionally, fluorescent (5) and chemiluminescent (6) tracers were prepared from (-)-acid (13) via in situ activation and subsequent reaction with 6-Fln-CH(2)NH(2) (17) or Acr-NH(2) (18) and hydrolysis. The key hapten (-)-acid (13) was prepared by quaternization of 3-hydroxypyridine derivative (S,S)-(-)-11 with iodide (S)-(-)-10 followed by selective hydrolysis. These immunreagents (immunogens 3 and 4 and tracers 5 and 6) are useful for the development of sensitive and high throughput immunoassays, such as FPIA and CLIA for Dpd (2).

Adjuvants, Immunologic↗
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Experimental febrile convulsions in epileptic chickens: the anticonvulsant effect of elevated gamma-aminobutyric acid concentrations.

The high seizure susceptibility in epileptic chickens is due to an autosomal recessive mutation. In 3-day-old chicks homozygous for the epilepsy gene (epileptics), elevation of body temperature using microwave diathermy evoked an initial febrile seizure resembling the clonic seizures evoked in epileptic chicks by photic stimulation. After complete recovery, this was followed by a clonic-tonic seizure. In nonepileptic heterozygote hatchmates (carriers) of the same age, only the latter seizure pattern was observed. In 16- to 17-day-old chicks of either phenotype, both seizure patterns were observed during hyperthermia. In all cases, the temperature at which seizures occurred was significantly lower in epileptic than in nonepileptic chicks, indicating a lower threshold for febrile seizures when there is an inherited predisposition to convulse. The occurrence of seizures was dependent on the body temperature and not on the rate of rise of temperature. Elevation of the brain gamma-aminobutyric acid (GABA) concentrations by administration of the GABA transaminase inhibitor gamma-vinyl GABA reduced the incidence of the initial febrile seizures and increased the latency in those birds that were not fully protected.

Aminocaproates↗
PubMedDate not supplied

Gene expression of atrial natriuretic factor in ovine fetal heart during development.

OBJECTIVE: The present study quantified the abundance of atrial natriuretic factor (ANF) messenger RNA (mRNA) and determined the developmental pattern of ANF gene expression in the four cardiac chambers of the ovine fetus during the last two-thirds of gestation. METHODS: Twenty-one fetuses from 13 time-dated pregnant ewes at gestational ages of 60-145 days were used for this study. Total RNA from fetal atria and ventricles was extracted and ANF mRNA was analyzed by Northern blotting. The ANF mRNA signal was quantified by light densitometry. The abundance of ANF mRNA in the cardiac chambers across gestational ages was analyzed by linear regression analysis and one-way analysis of variance. RESULTS: Atrial natriuretic factor mRNA was much more abundant in the atria than in the ventricles of all fetuses at each gestational age studied. Atrial ANF mRNA levels were lowest in the younger fetuses at 60 days and increased with advancing gestation. Ventricular ANF mRNA levels were highest in fetuses at 60 days and decreased to almost nondetectable levels near term. No difference in ANF mRNA abundance was noted between the right and left atria or the right and left ventricles at each gestational age. CONCLUSION: A developmental pattern of ANF gene expression is demonstrated in the ovine fetal heart during the last two-thirds of gestation. This pattern shows that atrial ANF mRNA abundance increases while ventricular abundance decreases as the fetus matures. Expression of the ANF gene in the fetal period may be regulated developmentally or induced by cardiovascular changes in utero.

Animals↗
PubMedDate not supplied

Ontogeny of insulin-like growth factor-I and -II gene expression in ovine fetal heart.

OBJECTIVE: Insulin-like growth factors (IGF)-I and -II have been implicated in growth and differentiation during embryonic and fetal development. To examine the role of the IGFs in growth of the fetal heart, we determined the gene expression of IGF-I and IGF-II in the four cardiac chambers of the ovine fetus from 58 to 146 days' gestation (term = 147 days). METHODS: Total RNA was obtained from the cardiac chambers, analyzed by Northern blot, and hybridized to ovine specific cDNA probes for IGF-I and IGF-II. The resulting autoradiograms were subjected to light densitometry, and the intensity of the IGF signals was normalized to the respective 28S ribosomal RNA signals. RESULTS: In the atria and ventricles, IGF-I mRNA abundance was very low throughout the gestational period studied, whereas IGF-II mRNA levels were higher and readily detectable. In the atria, IGF-I mRNA was very low at 60 days' gestation and appeared to increase gradually toward term. Abundance of IGF-II mRNA was high at 60 days, increased further until 120 days, and decreased slightly toward term. In contrast, in the ventricles, IGF-I mRNA increased from 60 to 100 days and then declined moderately at term. Levels of IGF-II mRNA in the ventricles were high at 60 days and decreased progressively to low levels at term. No difference in IGF-I or IGF-II mRNA levels was noted between the right and left atria or right and left ventricles. CONCLUSION: These results suggest that developmental patterns for IGF-I and IGF-II gene expression exist in the ovine fetal heart, and the patterns differ between the atria and ventricles. Further, these gestational trends differ from those for atrial natriuretic factor (ANF) found in our previous studies, indicating that expression of the ANF gene in the fetal heart may not be associated with cardiac growth and differentiation.

Animals↗