Simultaneous analysis of substrates,products, and inhibitors of xanthine oxidase by high-pressure liquid chromatography and gas chromatography

Procedures are presented for the simultaneous analysis of hypoxanthine, xanthine, allopurinol, oxipurinol, and uric acid in standard mixtures and physiological fluids using gas chromatography (gc) or high-pressure liquid chromatography (hplc). Excellent correlation was obtained between the two methods for hypoxanthine, xanthine, oxipurinol, and uric acid. There are advantages and disadvantages to both methods. hplc requires no prior derivatization, uses isocratic elution with a buffer containing no organic solvent, and has 50- to 100-fold greater sensitivity than gc. Simpler methods of prepurification, readily adapted to clinical laboratories, can be used for hplc analysis. Although substances that are found in some urine samples from cancer patients interfere with hplc, separations by gc are not affected by these substances.     

Evolution of haplodiploidy: Models for inbred and outbred systems

Several new models are proposed for the evolution of haplodiploidy. Each of these models is evaluated for its ability to explain (1) special problems associated with transition to haplodiploidy from a population of diplodiploid progenitors, (2) current patterns of population structure in haplodiploid and related species, and (3) the evolution of genetic systems similar but not identical to haplodiploid systems. Of the new models, three are based on special conditions associated with inbreeding. Close inbreeding provides for the automatic effects of reduced problems in expressing recessives, lowered differences in gain from heterozygosity (to produce both heterotic effects and a greater variety of offspring) between haploid and diploid males, effective protection of haploids from direct competition with diploids, and a mechanism for the spread of haplodiploidy through gains derived from increased ability to control sex ratio. These models differ in the context where gain from sex ratio control is expressed. Pathways for the evolution of haplodiploidy in outbreeding populations are also discussed. Females who parthenogenetically produce haploid males have high genetic relatedness to their sons. If the sperm of these males is used to make both sons and daughters, i.e., through matings with diplodiploid females, there may be a net gain for haplodiploids. Another outbreeding model, modified from S. W. Brown (1964, Genetics49, 797–817), deals with selection for females producing haploid males in populations where there are driving sex chromosomes. Biases created by drive in sex ratio may allow haplodiploid females to be the only effective producers of males in the population. Several of the new models explain the whole range of haplodiploid and related adaptations and provide predictions that appear to be more consistent with the known structure of contemporary populations than those available in current models.     

Malate decarboxylation in isolated mitochondria from the crassulacean acid metabolism plant Sedum praealtum

Mitochondria isolated from the Crassulacean acid metabolism plant Sedum praealtum were demonstrated to decarboxylate added malate at basal rates of 30–50 μmol mg^?1 original chlorophyll h^?1. The basal rate could be stimulated markedly by the addition of ADP, oxaloacetic acid, an uncoupler of oxidative phosphorylation, or NAD, with maximum rates of 70–100 μmol mg^?1 original chlorophyll h^?1 observed. These observed rates were high enough to account for a large proportion of the estimated rate of malate decarboxylation in vivo. The major products of malate oxidation by the mitochondria in most cases were found to be pyruvate and CO₂, indicating that malate oxidation in these mitochondria proceeds mainly through NAD malic enzyme rather than NAD malate dehydrogenase. Under conditions employed little of the pyruvate formed was further oxidized, suggesting a fate other than oxidation (conversion to starch) for this pyruvate. Malate decarboxylation by mitochondria and by partially purified NAD malic enzyme was markedly inhibited by NaHCO₃. A possible physiological role is suggested for this inhibition as a feedback control on the enzyme.     

Oxidative demethylation of t-butyl alcohol by rat liver microsomes

Tertiary butyl alcohol has often been used experimentally as a “non-metabolizable” alcohol. In this report, evidence is presented that t-butanol serves as a substrate for rat liver microsomes and that it is oxidatively demethylated to yield formaldehyde. The apparent K_m for t-butanol is 30 mM while V_max is about 5.5 nmol per min per mg microsomal protein. Formaldehyde production is stimulated by azide, which prevents destruction of H₂O₂ by catalase. Hydroxyl radical scavenging agents, such as benzoate, mannitol, and 2-keto-4-thiomethylbutyrate, suppress formaldehyde production. Therefore, the microsomal reaction pathway appears to involve the interaction of t-butanol with hydroxyl radicals generated from H₂O₂ by the microsomes. Formaldehyde is also produced when t-butanol is incubated with model hydroxyl radical-generating systems such as the iron-EDTA-stimulated oxidation of xanthine by xanthine oxidase or the iron-EDTA-catalyzed autoxidation of ascorbate. These results indicate that t-butanol cannot be used to distinguish metabolically-linked from non-metabolically-linked actions of ethanol.     

Mice with Spontaneous Mammary Tumors Develop Type-Specific Neutralizing and Cytotoxic Antibodies Against the Mouse Mammary Tumor Virus Envelope Protein gp52

Sera from C3H mammary tumor-bearing mice contain cytotoxic antibodies for mouse mammary tumor virus (MMTV)-producing cells, based on (51)Cr release in a complement-dependent serum cytotoxicity assay. The cytotoxic antibodies could be absorbed by purified C3H MMTV gp52 and C3H MMTV-infected cat cells (C3H [MMTV] CrFK) containing cell surface MMTV gp52. However, purified MMTV p27 and uninfected CrFK cat cells were negative. Absorption of the sera with GR (MMTV) CrFK cells also removed all of the cytotoxicity, whereas absorption with RIII (MMTV) CrFK cells was negative, even though all three infected cat cells contained equivalent amounts of gp52. The same C3H cytotoxic sera also neutralized the focus-forming capacity of a C3H MMTV pseudotype of Kirsten sarcoma virus containing MMTV gp52. In contrast, sera from mammary tumor-bearing GR and RIII mice did not neutralize the pseudotype. Furthermore, neutralization could be achieved only by anti-gp52 but not by anti-gp36, -p27, -p14, or -p10 C3H MMTV sera. The gp52's of C3H, GR, and RIII MMTV could also be distinguished by using a type-specific competition radioimmunoassay employing (125)I-gp52 of C3H MMTV and a hyperimmune rabbit anti-C3H MMTV serum. To demonstrate these differences directly, we studied the primary structure of gp52 on the surface of the C3H, GR, and RIII (MMTV) CrFK cells. Two-dimensional tryptic peptide maps of the cell surface lactoper-oxidase-catalyzed iodinated gp52's revealed a greater number of peptides common to the gp52's of C3H and GR MMTVs than to RIII MMTV gp52. These results demonstrate that gp52 is a major target antigen for both cytotoxic and neutralizing antibodies, that the cell surface and virion-associated gp52's of C3H, GR, and RIII MMTV contain both group- and type-specific determinants, and that C3H and GR MMTV gp52's are antigenically more related to each other than to RIII MMTV gp52. Furthermore, C3H mammary tumor-bearing mice develop type-specific antibodies capable of recognizing unique gp52 determinants and, therefore, are able to distinguish the gp52 of C3H MMTV from the gp52's of GR and RIII MMTV.     

Cross-reactivity between human and murine lymphocyte antigens

The anti-H-2 alloantiserum D-32 [(BlO.A(2R) × C3H.SW) anti-C3H] is cytolytic to human lymphocytes. Fab₂ blocking assays, indirect immunoprecipitation and sequential immunoprecipitation experiments showed that the anti-H-2 alloantiserum D-32 recognizes antigenic determinants which are expressed on the heavy chain of subpopulations of HLA-A, B antigens. These determinants are different from those defining the serological polymorphism of the HLA-A, B, C system, are the same as or spatially close to those recognized by the anti-HLA-A, B monoclonal antibody Q6/64 and are expressed on rabbit, rat or guinea pig lymphocytes.     

Modelling the rate of transfer of uranyl ions onto microbial cells

It has been observed that microbial cells can adsorb uranium ions from dilute aqueous solutions. Data collected from such experiments can be used to estimate correlative mass transfer coefficients. Physical observations bear out several inadequacies, however, of using an adsorption mass transfer model with a constant transfer coefficient relating the rate of transfer to the concentration gradient. By itself, the mass transfer model contains no provision to include (1) the initial transient, (2) the curvature in the later time rate curve, and (3) the non-linear curve relating initial levels of uranium concentration in solution to final residual uranium concentration for a set of batch experiments. It is found that a better match to observed data can be achieved by utilizing an intermediate state adsorption model analogous to a kinetic model based on an enzyme - substrate coupling scheme.     

Oxygen consumption during the fenton-type reaction between hydrogen peroxide and a ferrous-chelate (Fe2+-DTPA)

The Fenton-type reaction between ferrous diethylenetriamine pentaacetic acid (Fe²⁺-DTPA, 50–200 μM) and H₂O₂ (20–1000 μM) in phosphate buffer at pH 7.0 results in consumption of dissolved oxygen. This observation differs from many prior reports that oxygen is liberated when more concentrated solutions of H₂O₂ are decomposed by iron salts. The rate and total quantity of oxygen consumed were dependent upon the concentrations of ferrous chelate, H₂O₂, and excess DTPA. Evidence is provided that both the ferrous-DTPA chelate and free DTPA can participate in the oxygen-consuming reactions. Oxygen was also consumed during the Fenton reaction between ferrous ions and H₂O₂ when DTPA and phosphate buffer were omitted. Under these conditions, oxygen evolution was observed at higher H₂O₂ concentrations (e.g., 400 μM). The consumption of oxygen during the Fenton-type reaction of an iron chelate at neutral pH may be relavant to events that take place in biologic systems.     

Determination of indoles and catechols in rat brain and pineal using liquid chromatography with fluorometric and amperometric detection

Tryptophan, serotonin, 5-hydroxyindoleacetic acid, and homovanillic acid were determined in rat brain by the direct injection of a centrifuged tissue homogenate into a liquid chromatographic—fluorometric/amperometric system. The above indoles, along with melatonin, were also determined in single rat pineal glands. The utility of the system in determining several additional catechols and indoles in brain was examined.     

The hypothalamo-choroidal tract

Summary Light and electron microscopic examination of choroid plexuses from lateral ventricles of water-deprived and subcutaneously or intravenously vasopressin administered rats reveal morphologic changes typical for vasopressin responsive fluid transporting epithelia during hormonal stimulation. Ultrastructural changes noted in both dehydrated and vasopressin treated animals included: the frequent occurrence of choroidal dark cells, dilatation of the lateral and basal intercellular spaces, increased vacuolization of the apical cytoplasm, and a change in microvillar conformation from the normal clavate type to those with a filiform shape. On the basis of the ultrastructural changes observed it is proposed that the choroid plexus be regarded as a target tissue for vasopressin. These findings indicated that a vasopressinmediated transchoroidal cerebrospinal fluid absorption capability exists.Supported by U.S.P.H.S. Grant HD-08867This work submitted as partial requirement for the Master of Science degree in the Department of Anatomy, Colorado State University