A new homogeneous assay system for specific nucleic acid sequences: poly-dA and poly-A detection.
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Biomedical subjects
Publications and source records attributed to R L Christensen.
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Four carotenoids, 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene and spheroidene, have been incorporated into the B850 light-harvesting complex of the carotenoidless mutant, photosynthetic bacterium, Rhodobacter sphaeroides R-26.1. The extent of pi-electron conjugation in these molecules increases from 7 to 10 carbon-carbon double bonds. Carotenoid-to-bacteriochlorophyll singlet state energy transfer efficiencies were measured using steady-state fluorescence excitation spectroscopy to be 54 +/- 2%, 66 +/- 4%, 71 +/- 6% and 56 +/- 3% for the carotenoid series. These results are discussed with respect to the position of the energy levels and the magnitude of spectral overlap between the S1 (2(1)Ag) state emission from the isolated carotenoids and the bacteriochlorophyll absorption of the native complex. These studies provide a systematic approach to exploring the effect of excited state energies, spectral overlap and excited state lifetimes on the efficiencies of carotenoid-to-bacteriochlorophyll singlet energy transfer in photosynthetic systems.
Four all-trans carotenoids, spheroidene, 3,4-dihydrospheroidene, 3,4,5,6-tetrahydrospheroidene, and 3,4,7,8-tetrahydrospheroidene, have been purified using HPLC techniques and analyzed using absorption, fluorescence and fluorescence excitation spectroscopy of room temperature solutions. This series of molecules, for which the extent of pi-electron conjugation decreases from 10 to seven carbon-carbon double bonds, exhibits a systematic crossover from S2----S0 (1(1)Bu----1(1)Ag) to S1----S0 (2(1)Ag----1(1)Ag) emission with decreasing chain length. Extrapolation of the S1----S0 transition energies indicates that the 2(1)Ag states of longer carotenoids have considerably lower energies than previously thought. The energies of the S1 states of spheroidenes and other long carotenoids are correlated with the S1 energies of their chlorophyll partners in antenna complexes of photosynthetic systems. Implications for energy transfer in photosynthetic antenna are discussed.
The role of protein kinase C in the mechanism of stimulation of glucose transport in rat adipocytes was investigated. Glucose transport was stimulated by dioleoylglycerol (DOG), tetradecanoyl phorbol acetate (TPA) and phospholipase C (PLC). Agents that inhibit protein kinase C (polymyxin B, gossypol and quercitin) also inhibited glucose transport that had been stimulated by DOG, TPA, PLC and insulin.
Measurements of protein-to-creatinine ratios in single-voided urine samples were compared with 24-hour urinary protein excretions for quantitation of proteinuria in inpatients and outpatients. Patients included those representing a broad spectrum of renal diseases, a wide range of proteinuria, and various degrees of reduction in glomerular filtration rate. Protein-to-creatinine ratios in single-voided urine samples correlated well with measurements of 24-hour urinary protein. This simple single-voided test is reliable and useful in the screening, assessment, and follow-up of proteinuria and avoids the problems associated with 24-hour urine collection.
We evaluated and compared three different commercial kit immunoassays for alpha-fetoprotein (AFP) before we implemented our neural tube defect screening program. Each kit can be used with either serum or amniotic fluid. Analytical recovery ranges for AFP reference sera within each kit's standard curve limits (in kilo-int. units/L) were 97-108% (7.5-180) for the Kallestad kit, 77-101% (21.8-436) for Amersham, and 92-100% (0-177) for Hybritech. CVs, within each manufacturer's standard-curve limits, for combined intra-assay (amniotic fluid pools) and inter-assay (kit serum controls) averaged 3.6-7.3% (Kallestad), 2.4-9.3% (Amersham (y) kit results showed a correlation of r = 0.97, y = 1.05x + 5.5 kilo-int. units per liter of maternal serum (n = 66; range, 2.0-98.5). Gestational age did not influence these assay correlations. The Kallestad AFP assay demonstrated a maternal serum positivity rate of 2.9% at greater than or equal to 2.5 (n = 655) and 8.9% at less than 0.5 (n = 423) multiples of the median. All kits performed well analytically.
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It has become apparent that mild factor deficiency states (i.e., Factor VIII, IX, and von Willebrand's syndrome) are frequent causes of postoperative bleeding and hemorrhagic complications following trauma. Despite their incidence, they frequently are not diagnosed or in some instances are misdiagnosed. In an attempt to improve the level of performance of factor assays, the College of American Pathologists recently has introduced factor-deficient plasmas into their proficiency testing program. Initially, plasmas deficient in Factor VIII were evaluated and more recently, plasmas deficient in Factors IX and XI were included. The results of the initial survey programs indicated a lack of precision and accuracy of Factor VIII and IX assays. Similar difficulties also were encountered with plasmas deficient in Factor XI. Extensive information also was gathered on the fundamentals of laboratory performance of factor assays. This information included data regarding preparation of the standard activity curves, source of deficient substrate plasmas, number of patient sample dilutions, etc. Also, it was evident that various thromboplastins have different degrees of sensitivity to the various deficiency states. A portion of this variability may be attributable to the choice of activator, however, it would appear that the phospholipid content of the partial thromboplastins is of equal importance.
Glutamic acid decarboxylase (GAD) from Neurospora crassa was assayed in dormant and germinating conidia that had been permeabilized by toluene and methanol. N. crassa conidia contained 10 times the GAD activity found in vegetativemycelia. During conidial germination, GAD activity rapidly decreased to low levels before germ tubes appeared. GAD activity in germinating conidia closely followed the decreasing rate of glutamic acid metabolism. Inhibiting protein synthesis partially blocked the decrease in GAD activity, but eliminating exogenous carbon sources did not alter the initial rate of decrease in this enzyme. However, when conidia were incubated for more than 3 h in distilled water, GAD activity began to increase and eventually reached levels comparable to those in dormant conidia. Either GAD was reversibly inactivated or this enzyme could be synthesized from endogenous storage compounds when conidia were incubated in distilled water. These results are consistent with the hypothesis that GAD is a developmentally regulated enzyme that is responsible for catalyzing the first step in the metabolism of the large pool of free glutamic acid during conidial germination.
Granulocytes engaged in the phagocytosis of opsonized zymosan emit light by a process that is inhibited by superoxide dismutase and catalase. In the present report is is shown that light emission is the result of reactions between certain unspecified constituents of the ingested particles and some or all of the oxidizing agents (H2O2, O2),and possibly the hydroxyl radical and singlet oxygen) produced by the activated cells. This conclusion is based on a study of light emission by both activated cells ans artificial O2 generating system containing xanthine oxidase and purine. With these two systems light production required the presence of both zymosan and oxidizing agent, suggesting that the oxidation of particle components is necessary for luminescence to occur. The characteristics of the emission spectrum as well as the finding that granulocytes activated by a nonparticulate agent (F-) fail to liminesce show that light emission by the relaxation of singlet oxygen to the ground state does not contribute in a major way to the chemiluminescence of phagocytosing granulocytes; whether singlet oxygen contributes to chemiluminescence in other ways cannot be decided from the data available. Inasmuch as the oxidation of constituents of ingested particles is an important bacterial killing mechanism in the granulocyte, chemiluminescence may be viewed as a manifestation of the microbicidal activity of the cell.
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