Adenosine analogs with covalently attached lipids have enhanced potency at A1-adenosine receptors

Chemically functionalized congeners of N6-phenyladenosine and 1,3-dipropyl-8-phenylxanthine have been covalently coupled to fatty acids, diglycerides, and a phospholipid. The lipid-drug conjugates inhibit R-[3H]-phenylisopropyladenosine binding to A1-adenosine receptors in rat cerebral cortex membranes. A xanthine-phosphatidylethanolamine conjugate bound with a Ki value of 19 nM. Various xanthine esters of low potency are potential prodrugs. Amides of an adenosine amine congener (ADAC) with 18-carbon fatty acids exhibited Ki values at A1-adenosine receptors of 70 pM, representing a 130-fold enhancement over the affinity of the corresponding acetyl amide. The very high affinity of adenosine-lipid conjugates may be due to stabilization of these adducts in the phospholipid microenvironment of the receptor protein.     

Biosynthesis of squalene and sterols by rat aorta

The synthesis of nonsaponifiable compounds from radioactive mevalonate by segments of adult rat aorta was studied in vitro. The labeled products consisted largely of substances with the chromatographic and chemical behavior of squalene, lanosterol, lathosterol, and cholesterol. Even after 3 or 4 hr of incubation, the incorporation of mevalonate into squalene was higher than its incorporation into C(27) sterols; cholesterol contained less than 20% of the radioactivity in the total sterols. Lanosterol was the most highly labeled sterol. The level of radioactivity in lathosterol was comparable to the level in cholesterol. Small amounts of radioactivity were found in other sterols. Material with the same mobility on TLC as 7-dehydrocholesterol had less radioactivity than cholesterol, but more than sterols with the mobility of desmosterol. The results of measurements made after short periods of incubation showed that squalene and lanosterol became labeled before the other nonsaponifiable compounds.     

Protein and Ribonucleic Acid Synthesis during Germination of Uredospores

Existing evidence suggests that there is no net synthesis of protein during germination of spores of some obligate parasites. The problem was reinvestigated with enzymatic hydrolysis, rather than acid hydrolysis or solubility properties, to estimate changes in protein and RNA. Procedures are detailed for the use of uniformly labeled ¹⁴C-uredospores for the isolation and quantitative assay of amino acids and nucleosides resulting from hydrolysis.     

Assay and partial purification of epoxide hydrase from rat liver microsomes

Solubilized cytochrome P-450 monooxygenase and epoxide hydrase activities from rat liver microsomes have been separated by column chromatography. The highly active epoxide hydrase fraction is still contaminated with cytochrome P-450, which has very low monooxygenase activity. The highly purified cytochrome P-450 fraction possesses high monooxygenase activity and is essentially devoid of epoxide hydrase activity. Purification factors for the epoxide hydrase through four purification steps are similar with [³H]styrene oxide, [³H]naphthalene oxide, [³H]cyclohexene oxide, and benzene oxide as substrates. Failure of benzene oxide to inhibit hydration of styrene or naphthalene oxide in the most purified preparations in indicative of the presence of at least two hydrases. These purified cytochrome monooxygenase and hydrase preparations represent valuable tools for the study of the intermediacy of arene oxides in drug metabolism. Thus, with naphthalene, only naphthol is formed with the monooxygenase, while both naphthol and the dihydrodiol are formed in the presence of monooxygenase and hydrase. A convenient radiochemical synthesis of [³H]naphthalene 1,2-oxide and assays for the measurement of the hydration of [³H]naphthalene oxide and benzene oxide, based on differential extractions and high-pressure liquid chromatography, respectively, are described.     

Cell walls of germinating uredospores: I. Amino Acid and carbohydrate constituents

Synthesis of germ tube wall is a major quantitative event during germination penetration of fungi on host plants, but little is known of germ tube composition or metabolic regulation. Sonic oscillation was used to separate germ tubes from germinating uredospores of Uromyces phaseoli var. typica. Uniformly ¹⁴C-labeled wall fractions from both structures were prepared by repeated low speed centrifugation and extraction with polar and nonpolar solvents. Based on amino acid analysis, approximately 6 and 16% of the carbon from uredospore and germ tube walls, respectively, was present in amino acids readily accessible to protease. Covalent linkages between amino acid and carbohydrate of walls was indicated by analysis of fragments prepared by mild hydrolytic procedures and separated by column chromatography and paper electrophoresis. The existence of protein in wall structures may resolve some previous uncertainty about the occurrence of protein biosynthesis during germination of rust fungi. Glucose, mannose, and glucosamine were the only carbohydrate components identified in both germ tubes and uredospore walls but different percentages were observed (germ tubes 28: 16: 16; uredospore 6: 36: 6). In germ tubes, most of the glucosamine was present in linkages hydrolyzed only by strong acid treatment, suggesting chitin-like polymers. In uredospore walls, glucosamine appears to be associated with red uredospore pigment which has properties similar to those of a melanin. Approximately 20% of the carbon in walls could not be identified with known compounds, partially because of degradation during the analytical procedures.     

Cell Walls of Germinating Uredospores: II. Carbohydrate Polymers

Polymeric carbohydrates in ¹⁴C-labeled germ tube and uredospore walls of Uromyces phaseoli var. typica were studied by permethylation and by enzymatic hydrolysis. The native structure of the uredospore wall limited the effectiveness of both techniques with this wall, but evidence for two distinct polysaccharides was obtained. A linear (1→3) glucan, containing minor quantities of (1→6) linkages, may account for most of the glucose in the uredospore wall. A second uredospore polymer was a glucomannan similar to one reported for other rust fungi in that it consisted of approximately equal numbers of β(1→3) and β(1→4) mannosidic linkages with glucose as a minor component at the nonreducing end. Branching, most likely by (1→6) mannose links, was low. In contrast to uredospore wall, considerably more germ tube polysaccharide was accessible to enzymes and to methylation. Methylation studies indicate that (1→3) glucose and mannose bonds occur predominantly. Evidence from hydrolysis with exo- (β)-(1→3) glucanase suggests distinct wall regions of β(1→3) glycan, highly branched by (1→6) bonds, as well as wall regions of a glucomannan with alternating (1→3) glucose and (1→3) mannose residues. Polymer heterogeneity was indicated by differences in the proportions of mannose, glucose, and galactose as reducing end groups in different solubility fractions. In germ tube walls, but not in uredospore walls, glucosamine apparently existed as part of chitin polymer as evidenced by the isolation of N,N-diacetylchitobiose from chitinase digestion.     

Comparison of Activities of the Host-Specific Toxin of Helminthosporium maydis, Race T, and a Synthetic C(41) Analog

It previously had been proposed that the host-selective toxin of Helminthosporium maydis race T consists of a series of unusual linear (C₃₅ to C₄₅)polyketols, of equal toxicity on a weight or molar (10⁻⁸−10⁻⁹) basis. Previous laboratory synthesis of T-toxin analogs was limited to shorter (C₁₅ to C₂₆) versions which possessed the requisite specificity for susceptible corn (Zea mays) but were less toxic on a weight or molar (10⁻⁶−10⁻⁷) basis. In the present study, a C₄₁ analog with four β-ketol units spaced by CH₂ bridges as in native toxin has been synthesized. On a weight or molar basis, it is as effective as native toxin or its purified components in stimulating NADH oxidation of mitochondria from susceptible corn, thus providing firm evidence for the correctness of the proposed structures of T-toxin. Additional support derives from the observation that C₂₄ and C₂₆ analogs with -(CH₂)₄- and -(CH₂)₆- bridges between ketol groups are not as effective in stimulating NADH oxidation as are C₂₃ and C₂₅ analogs with the -(CH₂)₃- and -(CH₂)₅- bridges of native T-toxin.

It was calculated that a single molecule of the C₄₁ analog is at least 300 times more effective in stimulating mitochondrial oxidation than a molecule of the C₂₃ or C₂₅ analogs. This emphasizes the importance of chain length for toxicity, perhaps through perturbation of membrane functions of mitochondria and/or chloroplasts.

     

A machine for determining root length

Summary A machine for determining the root length of a sample is described. The machine is basically an opto-electronic scanner. Root segments are cut and placed in water on a glass plate (375×375 mm). The interruption of a light beam moving across the root sample is detected by a photo-diode and the total root length computed. Using this machine a root sample can be measured in less than 3 minutes. Detailed calibration was only conducted up to 50 m although samples as large as several hundred metres can be measured using this machine.The machine has a high degree of accuracy comparable with or better than other reported methods for determining root length.     

REGIONAL DISTRIBUTION AND PROPERTIES OF THE GLYCINE CLEAVAGE SYSTEM WITHIN THE CENTRAL NERVOUS SYSTEM OF THE RAT: EVIDENCE FOR AN ENDOGENOUS INHIBITOR DURING IN VITRO ASSAY

—The activity of the glycine cleavage system (GCS) was determined in homogenates from five specific regions of the rat CNS (telencephalon, midbrain, cerebellum, medulla-pons, and spinal cord). An inverse trend was noted between the glycine content and the specific activity of the GCS in the regions. A 25-fold range in the enzyme activities was found between the telencephalon (highest) and the spinal cord (lowest). The properties of the GCS activity in CNS homogenates agreed with those properties previously described for this system in partially purified preparations of liver and brain mitochondria (Kikuchi , 1973; Bruin et al., 1973). Within the CNS homogenates, the liberation of CO₂ from the carboxyl carbon of glycine was quantitatively coupled to the formation of serine. The presence of an endogenous inhibitor(s) within neural tissues was suggested by the non-additivity of the activities when homogenates from the various regions were combined. Moreover, homogenates of CNS tissue inhibited the GCS activity of liver homogenates, and an inverse relationship was found between the level of GCS activity in a given region of the CNS and its ability to inhibit the GCS activity of liver homogenates. This inhibition of liver activity was greatest when liver was incubated with homogenates of spinal cord (86%) and lowest when incubated with homogenates of telencephalon (20%). Because of this endogenous inhibition, the apparent activity of the GCS measured in vitro may not reflect the contribution of this enzyme system in the metabolism of glycine in vivo. Although the significance of this inhibition is not known, a possible role is discussed for the regulation of the levels in glycine and one-carbon pools within the CNS.