A radioimmunoassay for lithocholic acid conjugates in human serum and liver tissue

A sensitive and specific radioimmunoassay for glycine and taurine conjugates of lithocholic acid (CLCA) has been developed. ³H-glycolithocholic acid (S.A. = 17Ci/mmol) was used as tracer. Separation of free from antibody-bound bile acid was carried out using ammonium sulphate (saturated solution). The antiserum showed high specificity for both glyco and tauro conjugated lithocholate (100% cross reaction) and lithocholic acid (25% cross reaction). The sensitivity of the assay (1 pmole/tube), was adequate for measuring CLCA in peripheral blood and hepatic tissue in man.     

Synthesis and biological activity of amino acid conjugates of abscisic acid

We prepared 19 amino acid conjugates of the plant hormone abscisic acid (ABA) and investigated their biological activity, enzymatic hydrolysis by a recombinant Arabidopsis amidohydrolases GST-ILR1 and GST-IAR3, and metabolic fate in rice seedlings. Different sets of ABA-amino acids induced ABA-like responses in different plants. Some ABA-amino acids, including some that were active in bioassays, were hydrolyzed by recombinant Arabidopsis GST-IAR3, although GST-ILR1 did not show hydrolysis activity for any of the ABA-amino acids. ABA-L-Ala, which was active in all the bioassays, an Arabidopsis seed germination, spinach seed germination, and rice seedling elongation assays, except in a lettuce seed germination assay and was hydrolyzed by GST-IAR3, was hydrolyzed to free ABA in rice seedlings. These findings suggest that some plant amidohydrolases hydrolyze some ABA-amino acid conjugates. Because our study indicates the possibility that different plants have hydrolyzing activity toward different ABA-amino acids, an ABA-amino acid may function as a species-selective pro-hormone of ABA.     

Synthesis of dihydroquinopimaric acid conjugates with amino acids

Synthesis of dihydroquinopimaric acid amides and their 2β-succinyl and 2β-phthalyl derivatives containing residues of amino acids was carried out for the first time. Antiviral properties of the compounds synthesized were investigated.     

Study of prostaglandins and thromboxanes B2 using mass-spectrometry of secondary ions

Prostaglandins E, F, I2 and thromboxane B2 have been studied by secondary ion mass spectrometry. It is shown that the method is suitable for direct identification of these compounds either as free acids or as their sodium salts. The spectra of the former reveal their structural features, while with the latter information on the molecular weight can be obtained. The limit of detection (about 1 microgram) allows the analysis of prostaglandin solutions of 1 microgram/microliter concentrations used in pharmacological tests.     

Use of ion-exchange resins for removing prostaglandins from human urine prior to radioimmunoassay

Investigation of prostaglandin and prostaglandin metabolite levels in human urine by radioimmunoassay requires the removal of prostaglandins from urine in a predictable, reproducible manner. Screening of eighteen ion-exchange resins for their ability to remove tritiated prostaglandins from human urine was undertaken. Two resins, Amberlite IRP-64M and Amberlite CG-50 were found acceptable.     

Differential separation of thromboxanes from prostaglandins by one and two-dimensional thin layer chromatography

[¹⁴C]-labelled thromboxane B₂ and hydroxy fatty acids were isolated using thin layer and gas chromatographic procedures from human platelets incubated with [1-¹⁴C]-arachidonic acid. A number of TLC solvent systems were evaluated for differential separation of thromboxanes and hydroxy fatty acids from prostaglandins E₂, A₂, D₂ and F_2α. Chromatographic properties in nine different solvent systems are tabulated. Two dimensional TLC procedures suitable for complete resolution of mixtures of these compounds on a single plate were developed. The systems were used to demonstrate conversion of [1-¹⁴C]-arachidonic acid to thromboxane B₂ and prostaglandin E₂ by human lung fibroblasts in tissue culture.     

Synthesis of amino acid conjugates of 1,1′-dimethylferrocene: New chiral conjugates

Syntheses of diastereomeric mixtures of 1,1′-dimethylferrocene-2-Ala-OMe (3) and 1,1′-dimethylferrocene-3-Ala-OMe (4) are reported by peptide coupling of L-Ala-OMe to the enantiomeric mixtures of the corresponding acids, 1,1′-dimethylferrocene-2-carboxylic acid (1) and 1,1′-dimethylferrocene-3-carboxylic acid (2).     

Measurement of prostaglandins in embryonic tissue using radioimmunoassay

Although prostaglandins appear to play an important role in numerous physiological processes in the adult, neonate, and fetus, very little is known about the role of these compounds in the embryo. This study demonstrates that rat embryo homogenates synthesized 6-oxo-PGF_1α; PGE and PGF in markedly different amounts from endogenous substrate. Synthesis was inhibited by indomethacin (10 μM) in varying degrees (70–89%) depending on the prostaglandin. The metabolite of PGF_2α, 13,14-dihydro-15-keto PGF_2α (PGF_2α-M), was produced in limited amounts in the absence of exogenous NAD. In the presence of exogenous NAD and PGF however, embryonic homogenates produced PGF_2α-M. The potential role of prostaglandins during embryogenesis is discussed.     

Variables associated with radioimmunoassay of prostaglandins in plasma

This study examined a number of variables which influence radioimmunoassay measurements of prostaglandins in plasma. Comparative assays in specimens obtained from normal subjects and processed by different methods demonstrated that prostaglandin measurements were uniformly lower when blood was anticoagulated with EDTA, aspirin or indomethacin was added to inhibit further synthesis of prostaglandins , samples were centrifuged at 4°C at speeds which removed platelets from the sample, and assays were performed when plasma was thawed for the first time. Examination of assay results also revealed that the presence of unextracted plasma enhanced the binding of labeled ligands to antisera (%B_o) but thereafter, the displacement of ligand by increasing quantities of the reference prostaglandin paralleled the results in buffer throughout the range of the assay. In subsequent studies therefore the %B_o for plasma assays was adjusted on the basis of ligand binding in the presence of comparable volumes of pooled plasma from aspirin-treated subjects. Results of 7 prostaglandin assays in plasma and serum of normal subjects are reported.     

Synthesis of an asymmetrically substituted AZA crown ether as metal and amino acid binding site in DNA conjugates

Crown ether 4 as a receptor core for protonated primary amines such as amino acids has been synthesized and incorporated into oligodeoxynucleotides as dangling ends.     

Distribution of tritium in D-glucose and starch labeled by tritium-atom bombardment

Samples of D-glucose and starch were labeled by tritium-atom bombardment. Up to 51% incorporation into D-glucose as non-labile tritium was achieved for crystalline, anhydrous D-glucose and 41% for the amylose-butyl alcohol complex. Distribution of tritium in the carbon skeleton of D-glucose was calculated by comparing the specific molar activity of D-glucose with that of its derivatives. Derivatives prepared were D-gluconic acid, D-arabino-hexulose phenylosotriazole, 4-formyl-2-phenyltriazole, 2-phenyltriazole-4-carboxylic acid, D-arabino-hexulose phenylflavazole, 3-formyl-1-phenylflavazole, and formaldehyde dimedone. The tritium distribution showed definite structural effects. Generally, the products from films of D-glucose and the amylose-butyl alcohol complex had nearly uniform distribution of tritium in D-glucose. The product from crystalline α-D-glucose monohydrate had zero tritium at C-2 and twice the expected amount of tritium at C-5, and that from starch granules had zero or near zero tritium at C-3 and close to twice the expected amount of tritium at C-2.     

Solution conformation and electrostatic potential distribution of prostaglandins and thromboxanes and their relation to specificity

ProstaglandinThromboxaneSolution conformationElectrostatic potential     

Immunological and non-immunological synthesis and release of prostaglandins and thromboxanes from isolated guinea pig trachea

Specific radioimmunoassays were used to demonstrate the synthesis by the guinea pig trachea of 6-keto PGF_1α, TxB₂, and PGF_2α in addition to PGE₂. The rank order of both spontaneous and stimulated release was PGE₂ > PGF_2α > 6-keto PGF_1α = TxB₂. Ovalbumin-induced prostanoid release from sensitized tissue was antigen-specific. The release was unlikely to be a secondary consequence of tracheal contraction since incubations with calcium ionophore A23187, at a concentration which produces an equivalent magnitude of contraction of sensitized trachea, did not induce a significant PG or Tx production. In contrast, significantly higher prostanoid synthesis was induced by A23187 in unsensitized than sensitized trachea. Thus sensitization altered the profile of arachidonic acid metabolism evoked by the ionophore.     

Interaction of free indole-3-acetic acid and its amino acid conjugates in tomato hypocotyl cultures

The interaction of free IAA and its amino acid conjugates on growth and development of cultured tomato hypocotyl tissue (Lycopersicon esculentum Mill. cv. Marglobe) was studied. In a nutrient medium containing 10 mol/L of benzyladenine, free IAA stimulated shoot and root development with little callus proliferation. In contrast, all IAA-amino acid conjugates tested supported mostly callus growth. Simultaneous application of free IAA and its conjugates resulted in the expression of mixed morphogenetic responses (i.e., both vigorous callus growth and organogenesis resulted). Growth kinetics and the effect of temporal exposure of the tissues to the bound and the free auxin suggest that some IAA-amino acid conjugates may specifically influence plant morphogenesis in ways that cannot be easily explained as simply a function of their slow hydrolysis to release free IAA.Abbreviations IAA indole-3-acetic acid - IAA-Ala N-(indol-3-ylacetyl)-l-alanine - IAA-Asp N-(indol-3-ylacetyl)-dl-aspartic acid - IAA-Lys N -(indol-3-ylacetyl)-l-lysine - IAA-Orn N -(indol-3-ylacetyl)-l-ornithine - IAA-Thr N-(indol-3-ylaetyl)-l-threonine     

Use of phosphorus-32 labeled polyphosphoric acid for synthesis of labeled purine and pyrimidine 5'-ribonucleotides

Phosphorus-32 labeled polyphosphoric acid was used to phosphorylate the 2′,3′-O-isopropylidene derivaties of purine and pyrimidine ribonucleosides. The reaction mixture, which contained isopropylidene ribonucleoside 5′-phosphates, was subjected to selective hydrolysis, which removed the isopropylidene protecting group. The polyphosphate chains remained intact. Chromatography revealed the presence of ribonucleoside 5′-mono-, di-, tri-, tetra-, and higher phosphates. The distribution of radioactive label was established by liquid scintillation counting and enzyme assay. The triply labeled adenosine triphosphate prepared in this manner proved to be enzymically active and gave a positive firefly bioluminescence response.