Measurement of adrenolutin as an oxidation product of catecholamines in plasma

Using the reverse phase high-performance liquid chromatography (HPLC) with mobile phases composed of simple acids, we have developed an assay technique for the measurement of adrenolutin, one of the oxidation products of catecholamines, in rat plasma. Ion-pairing chromatography permits the separation and quantitation of plasma adrenolutin (M) in a linear manner. Sample preparation involved the precipitation of plasma proteins with perchloric acid and it is easier to handle a large number of samples at a time. However, we were unable to demonstrate the presence of adrenochrome, another oxidation product of catecholamines, in plasma since adrenochrome was rapidly destroyed in acid as well as in blood and was quickly changed, into adrenolutin. Adrenolutin peak in HPLC was confirmed by 1) the retention time; 2) co-injection of adrenolutin and; 3) the appearance of ³H-adrenolutin after injection of ³H-norepinephrine. Administration of different catecholamines as well as adrenochrome and adrenolutin in rats also increased the level of adrenolutin in plasma. Adrenolutin was found to be present in plasma in other species including dog, rabbit and pig. High level of adrenolutin, which may represent total concentration of aminolutin in plasma, suggests the presence of an efficient mechanism for the oxidation of catecholamines under in vivo conditions.     

The structure and evolution of the spider monkey delta-globin gene

We have isolated the delta-globin gene of the New-World spider monkey, Ateles geoffroyi, and compared its nucleotide sequence with those of other primate delta- and beta-globin genes. Among primate delta-globin genes, the rate of nonsynonymous substitutions is much less than the rate of synonymous substitutions. This suggests that primate delta- globin genes may remain under evolutionary conservation, perhaps because hemoglobin A2 has an as yet unknown physiological importance.      

Modulation of xanthine dehydrogenase and oxidase activities during the hormonal induction of vaginal epithelial differentiation in ovariectomized mice

In situ hybridization and immunocytochemical techniques have been used to examine the distribution of vitamin-D-induced calbindin mRNA and calbindin protein in enterocytes lining the crypts and villi of chicken small intestine. Basal villus enterocytes contained approximately twice as much calbindin but over three times as much calbindin mRNA compared to values found in basal crypt and upper villus enterocytes, all values being measured 2 days after vitamin D injection into D-deficient chickens. Virtually no calbindin mRNA was detected in tissues taken from control D-deficient birds. Direct proportionality found between calbindin mRNA and calbindin content in enterocytes of basal crypt, mid and upper villus suggests pre-translational control over calbindin synthesis. The implications of possible inefficient translation of calbindin mRNA in basal villus enterocytes are discussed. Present methods of analysis provide a novel way to study mechanisms controlling gene expression throughout the whole process of enterocyte differentiation.     

Cross-linking studies with the uvrA and uvrB proteins of E. coli

The interactions of the uvrA and uvrB proteins with DNA have been investigated using a DNA-protein cross-linking technique. It is demonstrated that hydrolysis of ATP by the uvrA protein facilitates cross-linking of this protein to single-stranded DNA, whether the DNA is UV irradiated or not. In contrast, cross-linking to unirradiated double-stranded DNA is not facilitated by ATP hydrolysis and is in fact increased by the substitution of the non-hydrolysable analogue aTP gamma S for ATP. In the presence of ATP, a dose-dependent increase is observed in the amount of uvrA protein which can be cross-linked to UV-irradiated double-stranded DNA. Binding of uvrB protein to puvrA-DNA complexes has a stabilising effect and increases the number of complexes which can be cross-linked whether the substrate is single- or double-stranded DNA. We can find no evidence that ATP hydrolysis by uvrA protein results in unwinding of UV-damaged DNA.     

Ethephon and auxin induce mycorrhiza-like changes in the morphology of root organ cultures of Mugo pine

The effects of ethylene and auxin on the morphology and anatomy of root organ cultures of Pinus mugo Turra var. mugo were investigated to test the hypothesis that changes in root morphology associated with formation of ectomycorrhizae may be related to ethylene produced by ectomycorrhizal fungi or by host plant roots in response to fungus-produced auxin. Morphological changes characteristic of mycorrhizal infection include dichotomous branching of lateral roots, inhibition of root hair formation and enlargement of cortical cells. Lateral roots on non-mycorrhizal root organ cultures, grown in a defined medium, underwent dichtotomous branching while root hair formation was inhibited in response to the ethylene released by 50 and 100 μ M ethephon (2-chloroethylphosphonic acid), but no effect on cortical cell dimensions was observed. The auxin, naphthaleneacetic acid (1 and 10 μ M ) also stimulated dichotomous branching and inhibited root hair formation, but to a lesser extent and with a greater lag time than ethephon. Auxin-stimulated ethylene production by root organ cultures was demonstrated. This appeared to be responsible, at least in part, for the auxin-induced dichotomous branching since the ethylene action inhibitor, silver thiosulfate (0.1 m M ) inhibited the response to auxin by 35%.     

Biochemical pathways in prokaryotes can be traced backward through evolutionary time

For the first time, a credible prokaryotic phylogenetic tree is being assembled by Woese and others using quantitative sequence analysis of oligonucleotides in the highly conservative rRNA. This provides an evolutionary scale against which the evolutionary steps that led to the arrangement and regulation of contemporary biochemical pathways can be measured. This paper presents an emerging evolutionary picture of aromatic amino acid biosynthesis within a large superfamily assemblage of prokaryotes that is sufficiently developed to illustrate a new perspective that will be applicable to many other biochemical pathways.      

Reactions of the UVRABC excision nuclease with DNA damaged by diamminedichloroplatinum(II).

Mutants of Escherichia coli, which are blocked in excision repair (uvrA6, uvrB5, or uvrC34) are exceptionally sensitive to the antitumor drug cis-Pt(II)(NH3)2Cl2 (cis-DDP) but not the trans isomer. Plasmid DNA, damaged by either the cis or trans compound and treated with the UVRABC excision nuclease was cut as shown by conversion of supercoiled DNA to relaxed forms. All three protein products of the uvrA, uvrB, and uvrC genes were required for incision. End-labeled fragments damaged with cis-DDP and reacted with the UVRABC nuclease were cut at the 8th phosphodiester bond 5' and at the 4th phosphodiester bond 3' to adjacent GG's. DNA treated with trans-DDP was not cut appreciably at adjacent GG's by the repair enzyme as subsequent analysis of reaction products after enzyme digestion gave a pattern similar to those obtained with control untreated fragments. The results indicate that the UVRABC nuclease may promote cell survival by the removal of adjacent GG's which are crosslinked by cis-Pt(II)(NH3)2Cl2.     

Radiosensitization, pharmacokinetics, and toxicity of a 2-nitroimidazole nucleoside (RA-263)

A 2-nitroimidazole nucleoside, 1-(2',3'-dideoxy-alpha-D-erythro-hex-2'-enopyranosyl)-2-nitroimida zole (RA-263), has been investigated for its radiosensitization, pharmacokinetics, and toxicity properties. The in vitro radiosensitization tests against hypoxic Chinese hamster (V-79) cells demonstrated that RA-263 was a more potent radiosensitizer than misonidazole and at 2 mM concentration approached the oxic curve. Significant in vitro radiosensitization activity was also observed in EMT6 mammary tumor cells. The in vitro cytotoxicity data suggested that RA-263 is considerably more toxic to hypoxic cells than misonidazole. The increased cytotoxicity may be related to its higher depletion of nonprotein thiols (NPSH) than misonidazole. The combined effects of radiosensitization and hypoxic cell toxicity were measured by preincubation of the V-79 cells for 4 h under hypoxic conditions before irradiation. The results demonstrated a synergistic response by causing a significant decrease in the extrapolation number with loss of shoulder of the radiation survival curves. The in vivo radiosensitization experiments conducted by the in vivo-in vitro cloning assay with the EMT6 mammary tumor indicate that RA-263 is an effective sensitizer. Pharmacokinetic data suggested that RA-263 was eliminated from plasma by a rapid alpha phase and a slower beta phase with T 1/2 of 36 and 72 min, respectively. The concentration in the brain was approximately one-sixth of tumor concentration, suggesting that RA-263 is excluded from the CNS. Moreover, RA-263 was two times less toxic than misonidazole on equimolar basis by acute LD50 tests. This agent was also significantly less mutagenic than misonidazole in a strain of Escherichia coli.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.