Functional regulation of xanthine oxidoreductase expression and localization in the mouse mammary gland: evidence of a role in lipid secretion.

Xanthine oxidoreductase (XOR), a key enzyme of purine metabolism, has been implicated in the secretion of the milk fat droplet in lactating mammary epithelial cells, possibly through structural interactions with other milk fat globule proteins including butyrophilin (Btn) and adipophilin (ADPH). To help determine the mechanism by which XOR is regulated, we examined the expression and localization of XOR in the non-secretory states of late pregnancy and induced involution compared with the state of active secretion. XOR mRNA levels started to increase at mid-pregnancy, turned sharply upwards at the onset of lactation and decreased rapidly with forced involution, indicating transcriptional control of the enzyme level by differentiation and secretory function. During pregnancy and involution the enzyme was diffusely distributed in the cytoplasm, but moved rapidly to the apical membrane of the cells when secretion was activated, where it colocalized with both Btn and ADPH, similar to the situation in the milk fat globule itself. Size-exclusion chromatography of solubilized milk fat globule membrane proteins showed that XOR formed a sulphydryl-bond-dependent complex with Btn and ADPH in the milk fat globule membrane. XOR returned to a diffuse cytoplasmic localization shortly after induced involution, while Btn remained localized to the apical membrane, suggesting that localization of XOR is not dependent on the presence of Btn in the apical membrane. Our findings indicate that the expression and membrane association of XOR in the mammary gland are tightly regulated by secretory activity, and suggest that the apical membrane association of XOR regulates the coupling of lipid droplets to the apical plasma membrane during milk lipid secretion.

The N-terminal region of human progesterone B-receptors: biophysical and biochemical comparison to A-receptors.

To understand the basis for functional differences between the two human progesterone receptors (PR), we have carried out a detailed biochemical and biophysical analysis of the N-terminal region of each isoform. Extending our previous work on the A-isoform (Bain, D. L, Franden, M. A., McManaman, J. L., Takimoto, G. S., and Horwitz, K. B. (2000) J. Biol. Chem. 275, 7313-7320), here we present studies on the N-terminal region of the B-isoform (NT-B) and compare its properties to its A-receptor counterpart (NT-A). As seen previously with NT-A, NT-B is quantitatively monomeric in solution, yet undergoes N-terminal-mediated assembly upon DNA binding. Limited proteolysis, microsequencing, and sedimentation analyses indicate that the B-isoform exists in a non-globular, extended conformation very similar to that of NT-A. Additionally, the 164 amino acids unique to the B-isoform (BUS) appear to be in a more extended conformation relative to sequences common to both receptors and do not exist as an independent structural domain. However, sedimentation studies of NT-A and NT-B show differences in the ensemble distribution of their conformational states. We hypothesize that isoform-specific functional differences are not due to structural differences, per se. Rather, the transcriptional element BUS, or possibly other transcription factors, causes a redistribution of the conformational ensemble by stabilizing a more functionally active set of conformations in NT-B.

The therapeutic potential of chemokine-toxin fusion proteins.

Activated cells of the immune system and the biochemical mediators they produce, underlie the pathology and secondary tissue damage in a wide range of diseases and traumas. One approach to therapy is to inhibit specific mediators in various biochemical pathways or cascades with the use of biological response modifiers. An alternative approach is to suppress or eradicate diseased cells or activated cells that fuel the disease and secondary tissue damage processes. Osprey has chosen to develop novel versatile cell-targeting agents that exploit the cell biology of the chemokine superfamily of receptors and ligands.

Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells.

The synthesis and secretion of lipids by mammary epithelial cells is a highly ordered process that involves several distinct steps. Triacylglycerols are synthesized in the endoplasmic reticulum and incorporated into microlipid droplets which coalesce into cytoplasmic lipid droplets. These are vectorially transported to the apical plasma membrane where they are secreted into the milk surrounded by a membrane bilayer. The origin of this membrane as well as the mechanism by which cytoplasmic lipid droplets form and become surrounded by membrane is poorly understood. Proteomic analysis of the protein composition of milk fat globules and cytoplasmic lipid droplet has revealed that the endoplasmic reticulum is not only involved in the synthesis of the lipid but also potentially contributes to the membrane component of milk fat globules. The proteins identified suggest possible mechanisms of multiple steps during this process. Completion of the proteome of milk fat globule membranes and cytoplasmic lipid droplets will provide the necessary reporter molecules to follow and dissect the mechanisms of the sorting and ultimate secretion of cytoplasmic lipid droplets.

Activation of the human aldehyde oxidase (hAOX1) promoter by tandem cooperative Sp1/Sp3 binding sites: identification of complex architecture in the hAOX upstream DNA that includes a proximal promoter, distal activation sites, and a silencer element.

Aldehyde oxidase (AOX) is a member of the molybdenum iron-sulfur flavoproteins and is of interest for its role in clinical drug metabolism and as a source of reactive oxygen species (ROS) potentially involved in human pathology. The ROS derived from AOX contribute significantly to alcohol-induced hepatotoxicity. Therefore, expression of AOX could determine both the susceptibility of certain cells and tissues to clinically important pharmacologic agents and the levels of ROS produced under certain pathophysiological conditions. Although some pharmacologic agents regulate AOX enzyme activity, very little is known about the activation or regulation of the human AOX gene (hAOX). In the present study, we sought to identify features in the upstream DNA of hAOX that could confer regulation of the gene, to locate and characterize the basal promoter apparatus activating hAOX, and to identify transcription factors that could mediate activation or regulation. We transfected promoter fusion constructs into epithelial cells from the lung and the mammary gland that express AOX in cell culture. The hAOX gene was found to possess a structurally complex region in the upstream DNA that contained sequences for a proximal promoter, enhancer sites, and silencer elements. In addition, we identified an essential role for the transcription factors Sp1 and Sp3 in the proximal promoter. Unexpectedly, hAOX was activated in lung and mammary epithelial cells by indistinguishable mechanisms. These observations reveal a potentially complex mode of hAOX gene expression in epithelial cells that is dependent on Spl and Sp3 transcription factors.

Biochemical characterization of a beta cell membrane fraction antigenic for autoreactive T cell clones.

The two NOD-derived T cell clones, BDC-2.5 and BDC-6.9, are CD4+, Vbeta4+, islet-specific, and diabetogenic. These two T cell clones show different response patterns to whole islet cell antigen, but were found to respond to the same fraction isolated from beta granule membranes. The clones were used to follow the antigenic activity in the biochemical purification of a beta cell membrane detergent lysate subjected to HPLC anion exchange (IEX) and size exclusion chromatography (SEC). Antigenic activity could be retained after lysis in only one detergent (octyl-beta-glucoside) among several tested. In order to detect solubilized antigen, beta membrane proteins were covalently linked to microlatex beads prior to being added to T cell proliferation assays, a technique that eliminated detergent toxicity and resulted in increased assay sensitivity. To purify the antigen, membrane proteins were absorbed onto an anion exchange column and after elution using a salt gradient, activity for the clones was found in a fraction containing 0.15-0.2 M NaCl. Subsequent analysis of this material by size exclusion chromatography provided an apparent molecular weight of the antigen to be between 50 and 80 kDa. Further attempts to purify the protein by SDS-PAGE resulted in loss of antigenic activity. It is possible that the elusive nature of this protein is a clue to its importance as an autoantigen.

The N-terminal region of the human progesterone A-receptor. Structural analysis and the influence of the DNA binding domain.

The role of the N-terminal region in nuclear receptor function was addressed by a biochemical and biophysical analysis of the progesterone receptor A-isoform lacking only the hormone binding domain (NT-A). Sedimentation studies demonstrate that NT-A is quantitatively monomeric, with a highly asymmetric shape. Contrary to dogma, the N-terminal region is structured as demonstrated by limited proteolysis. However, N-terminal structure is strongly stabilized by the DNA binding domain, possibly explaining the lack of structure seen in isolated activation domains. Upon DNA binding, NT-A undergoes N-terminal mediated assembly, suggestive of DNA-induced allostery, and consistent with changes in protease accessibility of sites outside the DNA binding domain. Microsequencing reveals that protease-accessible regions are limited to previously identified phosphorylation motifs and to functional domain boundaries.

Lactogenic hormones regulate xanthine oxidoreductase and beta-casein levels in mammary epithelial cells by distinct mechanisms.

Xanthine oxidoreductase (XOR) is a prominent component of the milk lipid globule, whose concentration is selectively increased in mammary epithelial cells during the transition from pregnancy to lactation. To understand how XOR expression is controlled in the mammary gland, we investigated its properties and regulation by lactogenic hormones in cultured HC11 mammary epithelial cells. XOR was purified as the NAD(+)-dependent dehydrogenase by benzamidine-Sepharose chromatography and was shown to be intact and to have biochemical properties similar to those of enzyme from other sources. Treating confluent HC11 cells with prolactin and cortisol produced a progressive, four- to fivefold, increase in XOR activity, while XOR activity in control cells remained constant. Elevated cellular XOR activity was correlated with increased XOR protein and was due to both increased synthesis and decreased degradation of XOR. Prolactin and cortisol increased XOR protein and mRNA in the presence of epidermal growth factor, which blocked the stimulation of beta-casein synthesis by these hormones. Further, hormonal stimulation of XOR was inhibited by genistein (a protein tyrosine kinase inhibitor) and by PD 98059 (a specific inhibitor of the MAP kinase cascade). These findings indicate that lactogenic hormones stimulate XOR and beta-casein expression via distinct pathways and suggest that a MAP kinase pathway mediates their effects on XOR. Our results provide evidence that lactogenic hormones regulate milk protein synthesis by multiple signaling pathways.

Mouse mammary gland xanthine oxidoreductase: purification, characterization, and regulation.

Xanthine oxidoreductase (XOR) has been purified from lactating mouse mammary tissue and its properties and developmental expression have been characterized. XOR was purified 80-fold in two steps using benzamidine-Sepharose affinity chromatography. The purified enzyme had a specific activity of 5.7 U/mg and an activity to flavin ratio of 192. SDS-polyacrylamide gel electrophoresis showed that it was composed of a single (150 kDa) band and N-terminal sequence analysis verified that it was intact mouse XOR. Isoelectric focusing showed that purified XOR was composed of three catalytically active, electrophoretic variants with pI values of 7.55, 7.65, and 7.70. The majority of the XOR activity in both pregnant and lactating mammary glands was shown to exist as NAD+-dependent dehydrogenase (XD form), while the enzyme in freshly obtained mouse milk exits as O2-dependent oxidase (XO form). The activity and protein levels of XOR selectively increased in mammary tissue during pregnancy and lactation. The time course of these increases was biphasic and correlated with the functional maturation of the mammary gland. These results indicate that XOR may have novel, mammary gland-specific functions, in addition to its role in purine metabolism.

Gabapentin for self-injurious behavior in Lesch-Nyhan syndrome.

Self-injurious behavior is a common clinical problem in children with Lesch-Nyhan syndrome, an X-linked disorder of purine metabolism. This behavior is not observed in other conditions associated with increased serum concentrations of uric acid, hypoxanthine, and xanthine. Various neurotransmitters appear to play a pivotal role in self-injurious behavior. The authors present a patient with Lesch-Nyhan syndrome, whose self-injurious behavior was effectively treated with gabapentin, and discuss possible mechanisms of action.

cDNA cloning, sequencing, and characterization of male and female rat liver aldehyde oxidase (rAOX1). Differences in redox status may distinguish male and female forms of hepatic APX.

Molecular characterization of male and female rat liver aldehyde oxidase is reported. As described for the mouse liver, male and female rat liver expressed kinetically distinct forms of aldehyde oxidase. Our data suggest that the two forms arise as a result of differences in redox state and are most simply explained by expression of a single gene encoding aldehyde oxidase in rats. In support of this argument we have sequenced cDNAs from male and female rat liver. We examined mRNA expression by Northern blot analysis with RNA from males and females, from several tissues, and following androgen induction. Purified rat liver enzyme from males or females revealed a single 150-kDa species consistent with cDNA sequence analysis. Both male and female forms were reactive to the same carboxyl-terminal directed antisera. Km(app) values obtained in crude extracts of male or female rat liver and post-benzamidine-purified aldehyde oxidase differed substantially from each other but could be interconverted by chemical reduction with dithiothreitol or oxidation with 4,4'-dithiodipyridine. Our data indicate that a single gene is most likely expressed in male or female rat liver and that the kinetic differences between male and female rat liver aldehyde oxidases are sensitive to redox manipulation.

Alcohol-induced breast cancer: a proposed mechanism.

Alcohol consumption increases the risk for breast cancer in women by still undefined means. Alcohol metabolism is known to produce reactive oxygen species (ROS), and breast cancer is associated with high levels of hydroxyl radical (*OH) modified DNA, point mutations, single strand nicks, and chromosome rearrangement. Furthermore, ROS modification of DNA can produce the mutations and DNA damage found in breast cancer. Alcohol dehydrogenase (ADH) and xanthine oxidoreductase (XOR) are expressed and regulated in breast tissues and aldehyde oxidase (AOX) may be present as well. Mammary gland XOR is an efficient source of ROS. Recently, hepatic XOR and AOX were found to generate ROS in two ways from alcohol metabolism: by acetaldehyde consumption and by the intrinsic NADH oxidase activity of both XOR and AOX. The data obtained suggests that: (1) expression of ADH and XOR or AOX in breast tissue provides the enzymes that generate ROS; (2) metabolism of alcohol produces acetaldehyde and NADH that can both be substrates for XOR or AOX and thereby result in ROS formation; and (3) ROS generated by XOR or AOX can induce the carcinogenic mutations and DNA damage found in breast cancer. Accumulation of iron coupled with diminished antioxidant defenses in breast tissue with advancing age provide additional support for this hypothesis because both result in elevated ROS damage that may exacerbate the risk for ROS-induced breast cancer.

Hypoxia regulates xanthine dehydrogenase activity at pre- and posttranslational levels.

Hypoxia increases the activity of xanthine oxidase (XO) and its precursor, xanthine dehydrogenase (XDH), but the mechanism of regulation is unclear. In hypoxic Swiss 3T3 cells, an early (0-24 h) cycloheximide-insensitive increase in XO-XDH activity, coupled with a lack of increase in de novo XO-XDH synthesis (immunoprecipitation) or mRNA levels (quantitative RT-PCR), demonstrated a posttranslational effect of hypoxia. Similarly, hyperoxia decreased XO-XDH activity faster than could be accounted for by cessation of XO-XDH protein synthesis. In further support of a posttranslational effect, cells transfected with a constitutively driven XDH construct displayed an exaggerated increase in activity in hypoxia but no increase in activity in hyperoxia. However, more prolonged exposure to hypoxia (24-48 h) induced an increase in XO-XDH mRNA levels and de novo XO-XDH protein synthesis, suggesting an additional pretranslational effect. Finally, hypoxic induction of XO-XDH activity was found to be cell-type-restricted. We conclude that control of XO-XDH levels by oxygen tension is a complex process which involves several points of regulation.

Cachectic effect of ciliary neurotrophic factor on innervated skeletal muscle.

Recombinant human ciliary neurotrophic factor (rh-CNTF) was reported to attenuate skeletal muscle wasting in rats after unilateral transection of the sciatic nerve (M. E. Helgren, S. P. Squinto, H. L. Davis, D. J. Parry, T. G. Bolton, C. S. Heck, Y. Zhu, G. D. Yancopoulos, R. M. Lindsay, and P. S. DiStefano. Cell 76: 493-504, 1994). Under the experimental conditions reported herein, the absolute masses of the denervated gastrocnemius and soleus muscles were not increased in mature or immature rats of either sex by treatment with rhCNTF. At the highest doses of rhCNTF (1 and 0.1 mg/kg), increases in the ratio of the masses of the denervated to the contralateral innervated gastrocnemius and soleus muscles could be attributed entirely to a muscle-wasting effect on the contralateral innervated muscle rather than any muscle-sparing effect on the denervated muscle. The muscle-wasting effects of rhCNTF were associated with reductions in body weight gain and reduced food intake. Pair-fed rats lost less body weight and skeletal muscle mass than rhCNTF-injected freely fed rats but experienced significantly greater loss of visceral mass. Male rats displayed greater loss of body weight and skeletal muscle mass than female rats. Recombinant inhibitors of the cachectic cytokines, tumor necrosis factor and interleukin-1, did not significantly alter the wasting effects of rhCNTF. These findings demonstrate that, in contrast to its well-characterized trophic effects on cells of the nervous system, rhCNTF causes atrophy of skeletal muscle by mechanisms involving both anorexia and cachexia based on the results of pair-feeding experiments.

Purification of rat liver xanthine oxidase and xanthine dehydrogenase by affinity chromatography on benzamidine-sepharose.

The oxidase form of xanthine dehydrogenase (XO; EC 1.1.3.22) has been purified approximately 200-fold from rat liver extracts using a three-step process of heat treatment, ammonium sulfate precipitation, and chromatography on benzamidine-Sepharose. The purified enzyme showed only minor contamination when analyzed by gel electrophoresis under either native or sodium dodecyl sulfate (SDS)-denatured conditions and appears to be intact based on its subunit size on SDS-polyacrylamide gel electrophoresis, its N-terminal amino acid sequence, and its ability to be converted to the NAD-dependent dehydrogenase form (XD; EC 1.1.1.204) by incubation with dithiothreitol. Isoelectric focusing analysis showed that the purified enzyme consists of two major, enzymatically active isoforms with average pI values of 6.13 and 6.23 and a minor enzymatically active isoform with an average pl value of 6.07. A similar purification of XD was achieved by preincubating the partially purified oxidase with dithiothreitol prior to affinity chromatography on benzamidine-Sepharose. The effects of benzamidine on the kinetic properties of purified rat XO were characterized at pH 8 and 9 and were compared to those of bovine milk XO. Benzamidine was found to be a weak competitive inhibitor of the purified rat enzyme with Ki values of 30 and 10 mM at pH 8 and 9, respectively. In contrast, the Ki values for benzamidine with bovine XO were more than 10-fold greater. The findings presented in this study show that benzamidine is a competitive inhibitor of XO and that affinity chromatography on benzamidine-Sepharose provides a simple, rapid, and effective means of purifying both the oxidase and dehydrogenase forms of rat XO.

Tyrosine kinase phosphorylation of GABAA receptors.

Phosphorylation of purified bovine brain GABAA receptors by the tyrosine kinase, pp60v-src was examined. pp60v-src phosphorylated two bands of 54-62 kDa and 48-51 kDa that migrated to approximately the same position as bands recognized by antisera against the beta 2 and gamma 2 GABAA receptor subunits, respectively. Bacterially expressed proteins containing the putative large cytoplasmic loops of the beta 1 and gamma 2L subunits were phosphorylated by pp60v-src, indicating that the phosphorylation sites are located in these subunit domains. The tyrosine kinase inhibitors, genistein and the tyrphostins B-42 and B-44, inhibited muscimol-stimulated 36Cl- uptake in mouse brain membrane vesicles (microsacs). magnitude of the tyrphostin B-44-induced inhibition of muscimol-stimulated 36Cl- uptake was significantly reduced in microsacs that were lysed and resealed under conditions that inhibit phosphorylation. GABA-gated Cl- currents were also inhibited by genistein and tyrphostin B-44 in Xenopus oocytes expressing alpha 1 beta 1 and alpha 1 beta 1 gamma 2L subunits. Consequently, protein tyrosine kinase-dependent phosphorylation appears to be another mechanism of regulating the function of GABAA receptors.

Regulation of tyrosine hydroxylase gene expression in IMR-32 neuroblastoma cells by basic fibroblast growth factor and ciliary neurotrophic factor.

The actions of basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) on tyrosine hydroxylase (TH) gene expression were studied using IMR-32 neuroblastoma cells. Treatment of these cells with bFGF for 3 days induced the expression of detectable levels of immunoreactive TH protein and TH mRNA. In contrast, CNTF did not affect TH expression unless bFGF was present. In the presence of saturating amounts of bFGF, CNTF increased TH protein and mRNA levels of TH two-to threefold over those found in bFGF-treated cultures. The effects of CNTF on TH expression diminished with increasing culture time, and after 6 days of incubation CNTF no longer enhanced TH levels. The requirement for bFGF as cofactor in the effects of CNTF on TH was specific, as CNTF did not affect TH when it was coadministered with 8-(4-chlorophenylthio)-cyclic AMP, another agent that stimulates TH development in this cell line, and bFGF was not required for CNTF to stimulate the development of choline acetyltransferase. Moreover, cotreatment with bFGF reduced the ability of CNTF to enhance choline acetyltransferase. These results demonstrate that bFGF and CNTF can enhance expression of TH and that bFGF can modify the effects of CNTF on neurotransmitter phenotype.

Identification of the candidate ALS2 gene at chromosome 2q33 as a human aldehyde oxidase gene.

Denver, Tokyo, and Salt Lake City investigators recently published different complimentary deoxyribonucleic acid (cDNA) sequences for human liver xanthine dehydrogenase/xanthine oxidase (XD/XO). The gene encoding the Denver cDNA was subsequently linked to juvenile familial amyotrophic lateral sclerosis (JFALS) at chromosome 2q33 and has been proposed as the ALS2 locus. The present investigation was undertaken to elucidate the differences between the three cDNA sequences, and we provide evidence that the Denver cDNA encodes aldehyde oxidase (AO): first, the Denver cDNA sequence diverged significantly from the Tokyo and Salt Lake City cDNA sequences which were very similar; second, the deduced protein sequence from the Denver cDNA was very similar to the amino acid sequence of purified rabbit liver AO protein; third, the deduced Denver protein sequence was 76% identical to the encoded 101 amino acid long peptides from partial cDNAs for rabbit and rat AO and 81.7% identical to 300 amino acids from an incomplete cDNA encoding bovine AO; fourth, the Denver gene was expressed in liver, kidney, lung, pancreas, prostate, testes, and ovary while the Tokyo XD gene was expressed predominantly in liver and small intestine; fifth, the Denver gene was previously mapped to chromosome 2q33 which is syntenic to the mouse AO locus on chromosome 1. Our results have revealed dramatic similarities in protein and DNA sequence in the human molybdenum hydroxylases, have uncovered unanticipated complexity in the human molybdenum hydroxylase genes, and advance the potential for AO derived oxygen radicals in JFALS and other human diseases.

Preparation and affinity purification of a novel, biologically active, CNTF fusion protein.

A biologically active fusion protein comprising a short hydrophilic leader peptide fused to the N-terminus of rat CNTF was generated using commercially available materials. The coding region for rat CNTF was sub-cloned into the pFLAG-1 vector and transfected into the JM 109 strain of E. coli. The transfected cells expressed high levels of the fusion protein (FLAG-CNTF) following induction by isopropyl beta-D-thiogalactoside (IPTG). FLAG-CNTF is expressed as insoluble material that was resolubilized by extraction with guanidine hydrochloride and purified by immuno-affinity chromatography. Analysis of the purified material by reverse phase HPLC and Western blot analysis indicated that FLAG-CNTF was composed of two closely related species that are greater than 99% pure after affinity chromatography. The purified FLAG-CNTF migrated as a 27 KD doublet on SDS polyacrylamide gel electrophoresis. Both bands of the doublet were shown to contain the FLAG peptide and CNTF by Western blot analysis, and amino acid sequence analysis demonstrated a single amino acid sequence corresponding to FLAG peptide and the N-terminus of CNTF. The purified fusion protein was tested for biological activity using the IMR-32 human neuroblastoma cell line. Treatment of IMR-32 cells with FLAG-CNTF increased the level of choline acetyltransferase (ChAT) in these cells 2-3-fold over that of control cells in a dose dependent manner. A direct comparison of the effects of FLAG-CNTF and recombinant human CNTF on IMR-32 ChAT activity showed that both factors exhibited similar potencies.(ABSTRACT TRUNCATED AT 250 WORDS)