GALT deficiency causes UDP-hexose deficit in human galactosemic cells

Previously we reported that stable transfection of human UDP-glucose pyrophosphorylase (hUGP2) rescued galactose-1-phosphate uridyltransferase (GALT)-deficient yeast from "galactose toxicity." Here we test in human cell lines the hypothesis that galactose toxicity was caused by excess accumulation of galactose-1-phosphate (Gal-1-P), inhibition of hUGP2, and UDP-hexose deficiency. We found that SV40-transformed fibroblasts derived from a galactosemic patient accumulated Gal-1-P from 1.2+/-0.4 to 5.2+/-0.5 mM and stopped growing when transferred from 0.1% glucose to 0.1% galactose. Control fibroblasts accumulated little Gal-1-P and continued to grow. The GALT-deficient cells had 157+/-10 micromoles UDP-glucose/100 g protein and 25+/-5 micromoles UDP-galactose/100 g protein when grown in 0.1% glucose. The control cells had 236+/-25 micromoles UDP- glucose/100 g protein and 82+/-10 micromoles UDP-galactose/100 g protein when grown in identical medium. When we transfected the GALT-deficient cells with either the hUGP2 or GALT gene, their UDP-glucose content increased to 305+/-28 micromoles/100 g protein (hUGP2-transfected) and 210+/-13 micromoles/100 g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75+/-12 micromoles/100 g protein (hUGP2-transfected) and 55+/-9 micromoles/100 g protein (GALT-transfected), respectively. Though the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2+/-0.02 mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1+/-0.4 mM). We found that 2.5 mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7 microM to 169 microM, without changes in apparent Vmax. The Ki of the reaction was 0.47 mM. Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase, which catalyzes the formation of UDP-N-acetylglucosamine. We conclude that intracellular concentrations of Gal-1-P found in classic galactosemia inhibit UDP-hexose pyrophosphorylases and reduce the intracellular concentrations of UDP-hexoses. Reduced Sambucus nigra agglutinin binding to glycoproteins isolated from cells with increased Gal-1-P is consistent with the resultant inhibition of glycoprotein glycosylation.     

Hyperoxic lung damage in mice: appearance and bioconversion of peptide leukotrienes

High concentrations of oxygen damage the lung and increase bronchoalveolar lavage (BAL) fluid levels of leukotrienes. We sought to identify the specific leukotrienes produced and their relationship to the severity of the lung damage and the inflammatory cell populations by exposing mice to 100% oxygen for up to 4 days. Leukotrienes were not detected in BAL fluid from air-exposed mice. Leukotriene D4 (LTD4) was found after 2 days of exposure to 100% oxygen, increased with longer periods of exposure, and then decreased while LTE4 appeared when the lung damage became severe. LTB4 and LTC4 were not found at any time. Neutropenic mice had identical results, indicating that neutrophils were not the source of the leukotrienes. To determine why LTC4 was not found and why LTD4 decreased and LTE4 increased on day 4, we measured the metabolic capacity of BAL supernatant for leukotrienes. Incubation of LTD4 in BAL supernatant from air-exposed mice resulted in the conversion of LTD4 to LTE4, which was blocked by L-cysteine, a dipeptidase inhibitor. Faster conversion occurred after exposure to 100% oxygen for 3 and 4 days. The rate of bioconversion correlated with the BAL protein concentration (r = 0.756, P less than 0.001), and it was similar in neutropenic and nonneutropenic mice. Little LTC4 and no LTE4 were converted in BAL supernatant from air- or oxygen-exposed mice. The early and progressive increase in LTD4 suggests that sulfidopeptide leukotrienes may play a role in the pathogenesis of hyperoxic lung damage. The increased dipeptidase activity during hyperoxic exposure may serve a protective role by converting the more potent LTD4 to the less potent LTE4.     

High-resolution radiation hybrid map of wheat chromosome 1D

Physical mapping methods that do not rely on meiotic recombination are necessary for complex polyploid genomes such as wheat (Triticum aestivum L.). This need is due to the uneven distribution of recombination and significant variation in genetic to physical distance ratios. One method that has proven valuable in a number of nonplant and plant systems is radiation hybrid (RH) mapping. This work presents, for the first time, a high-resolution radiation hybrid map of wheat chromosome 1D (D genome) in a tetraploid durum wheat (T. turgidum L., AB genomes) background. An RH panel of 87 lines was used to map 378 molecular markers, which detected 2312 chromosome breaks. The total map distance ranged from ~3,341 cR_35,000 for five major linkage groups to 11,773 cR_35,000 for a comprehensive map. The mapping resolution was estimated to be ~199 kb/break and provided the starting point for BAC contig alignment. To date, this is the highest resolution that has been obtained by plant RH mapping and serves as a first step for the development of RH resources in wheat.     

Explant culture of rat colon: A model system for studying metabolism of chemical carcinogens

Summary An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm² in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, β-retinyl acetate, and either 2.5% bovine albumin or 5% fetal bovine serum. The dishes were placed in a controlied-atmosphere chamber which was gassed with 95% O₂ and 5% CO₂. The chamber then was placed on a rocker platform which rocked at 10 cycles per min causing the medium to flow intermittently over the epithelial surface. The explants were incubated at 30°C. The viability of the tissue was measured both by incorporation of specific precursors into cellular macromolecules and by monitoring of tissue morphology with light and electron microscopy. Cultured rat colon was able to metabolize benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, aflatoxin B₁, dimethylnitrosamine, 1,2-dimethylhydrazine, and methylazoxymethanol acetate into chemical species that bind to cellular DNA and protein.     

Nicotine partially protects against paraquat-induced nigrostriatal damage in mice; link to alpha6beta2* nAChRs

Epidemiological studies indicate that smoking is a negative, and exposure to pesticides, a positive risk factor for Parkinson's disease (PD). The purpose of this study was to assess the interplay between these two factors in a rodent model of nigrostriatal damage. To approach this, mice were administered nicotine, the agent in smoke implicated in neuroprotection. They were then treated for 3 weeks with the pesticide, paraquat, while nicotine was continued. Paraquat treatment decreased (25%) nigral dopaminergic neurons, consistent with previous results. Chronic nicotine administration significantly protected against nigral cell damage, with only a 16% decline in mice treated with both nicotine and paraquat. Paraquat treatment also decreased (14%) the striatal dopamine transporter, an effect that was partially prevented by nicotine. These changes in the striatal dopamine transporter paralleled those in a select striatal alpha6beta2* nicotinic receptor (nAChR) subtype. In contrast, striatal alpha4beta2* nAChRs were not decreased with paraquat treatment, suggesting they are on a differential subset of dopaminergic terminals. The results show that nicotine treatment partially protects against paraquat-induced declines in nigrostriatal dopaminergic neurons to which a select population of alpha6beta2* nAChRs are localized. Moreover, these data support epidemiological findings that environmental influences can elicit opposing effects on nigrostriatal dopaminergic integrity.     

Retinal Muller glia initiate innate response to infectious stimuli via toll-like receptor signaling

Ocular surgeries and trauma predispose the eye to develop infectious endophthalmitis, which often leads to vision loss. The mechanisms of initiation of innate defense in this disease are not well understood but are presumed to involve retinal glial cells. We hypothesize that retinal Muller glia can recognize and respond to invading pathogens via TLRs, which are key regulators of the innate immune system. Using the mouse retinal sections, human retinal Muller cell line (MIO-M1), and primary mouse retinal Muller cells, we show that they express known human TLR1-10, adaptor molecules MyD88, TRIF, TRAM, and TRAF6, and co-receptors MD2 and CD14. Consistent with the gene expression, protein levels were also detected for the TLRs. Moreover, stimulation of the Muller glia with TLR 2, 3, 4, 5, 7 and 9 agonists resulted in an increased TLR expression as assayed by Western blot and flow cytometry. Furthermore, TLR agonists or live pathogen (S. aureus, P. aeruginosa, & C. albicans)-challenged Muller glia produced significantly higher levels of inflammatory mediators (TNF-α, IL-1β, IL-6 and IL-8), concomitantly with the activation of NF-κB, p38 and Erk signaling. This data suggests that Muller glia directly contributes to retinal innate defense by recognizing microbial patterns under infectious conditions; such as those in endophthalmitis.     

Comparison of metabolic rates and feed nutrient digestibility in conventional, genetically improved (GIFT) and genetically male (GMNT) Nile tilapia, Oreochromis niloticus (L.)

Various aspects of energy metabolism and feed digestibility were evaluated in two reportedly improved strains of Nile tilapia (Oreochromis niloticus) namely GIFT (genetically improved farmed tilapia) and GMNT (genetically male Nile tilapia) and compared with those of CNT (conventional Nile tilapia). Fish were stocked individually in a computer-controlled respirometer system at 27+/-0.1 degrees C for 10 weeks. Metabolic rates were measured at three different feeding levels: starved, maintenance (3.0 g kg(-0.8) day(-1)) and growth (7.5 g kg(-0.8) day(-1)) using a fishmeal based feed containing TiO2 marker (41% crude protein, 9% crude lipid and 19 kJ (g DM)(-1) gross energy). The standard metabolic rate (SMR), measured at the beginning of the experiment (45.4+/-4.6, 52.4+/-7.7 and 46.8+/-4.6 mg O2 kg(-0.8) h(-1) respectively for GIFT, GMNT and CNT), did not differ significantly between the groups (p<0.05). Similarly, non-significant differences were also observed in the routine metabolic rates under starved, maintenance and growth conditions but the variability was higher in the case of GMNT and CNT than in GIFT. The latter group showed a significantly lower active metabolic rate (145 mg O2 kg(-0.8) h(-1)) compared to GMNT and CNT (232 and 253 mg O2 kg(-0.8) h(-1), respectively) at maintenance feeding level. The specific dynamic action (% offered feed energy) showed no significant differences among the groups. Digestibility coefficients of feed dry matter, protein, lipid and energy for the three tilapia groups also did not differ significantly. Therefore, we concluded that the genetic improvement or modification in the GIFT or GMNT might not upgrade the inherent physiological potential compared to CNT as far as energy metabolism and digestion efficiencies are concerned.     

8-Substituted derivatives of adenosine 3',5'-cyclic phosphate require an unsubstituted 2'-hydroxyl group in the ribo configuration for biological activity.

Derivatives of adenosine 3',5'-cyclic phosphate (cAMP) with modifications in both the 2' and the 8 positions were synthesized and their enzymic activities as activators of cAMP-dependent protein kinase and as substrates for and inhibitors of cAMP phosphodiesterases were determined. Three types of derivatives were investigated: 8-substituted derivatives of O2'-Bt-cAMP, 8-substituted derivatives of 9-beta-D-arabinofuranosyladenine 3',5'-cyclic phosphate (ara-cAMP), and 8-substituted derivatives of 8,2'-anhydro-9-beta-D-arabinofuranosyladenine 3,'5'-cyclic phosphate (8,2'-anhydro-cAMP). The 8-substituted O2'-Bt-cAMP derivatives were synthesized by acylation of the preformed 8-substituted cAMP (8-HS-cAMP, 8-MeS-cAMP, and 8-PhCH2S-cAMP). 8-Br-O2'-tosyl-cAMP was sued as an intermediate for the preparation of 8,2'-anhydro-cAMP derivatives (8-HO-, 8-SH-, 8-H2N-, and 8-H3 CHN derivatives of 8,2'-anhydro-cAMP). 8-Substituted ara-cAMP derivatives were obtained by ring opening of 8-HO-8,2'-anhydro-cAMP with H+/H2O, NH3/MeOH, or MeONa/MeOH (to yield the 8-HO-, 8-H2N-, and 8-MeO-ara-cAMP derivatives). All of these doubly modified derivatives of cAMP are less than one-hundredth as active as cAMP at activating protein kinase and did not serve as substrates for the phosphodiesterase. These data show that the general inactivity of 2' derivatives of cAMP with kinase was not overcome by addition of an 8-substituent, even though many 8-substituted derivatives of cAMP activate the kinase more efficiently than does cAMP itself. In addition they show that while 2'-modification were tolerated by the phosphodiesterase, addition of an 8-substituent countermanded the allowable 2'-modification. The 8-substituted derivates of 02'-Bt-cAMP were found in general to be slightly better inhibitors of phosphodiesterase than the parent compounds containing no o2'-Bt substitution. As a group, the 8-substituted ara-cAMP derivatives were poorer inhibitors of phosphodiesterase than 8-substituted cAMP derivatives while the 8,2'-anhydro-cAMP derivatives were much poorer inhibitors than the 8-substituted ara-cAMP derivatives.     

Identification of an epitope within human integrin alpha 6 subunit for the binding of autoantibody and its role in basement membrane separation in oral pemphigoid

Oral pemphigoid (OP) is a rare chronic autoimmune disease characterized by blisters and erosive lesions in the oral mucosa. We identified an epitope for the binding of OP autoantibodies within the integrin alpha6 subunit, by cloning four overlapping fragments (A, B, C, and D). Immunoperoxidase studies demonstrated that all of the fragments were present in the oral mucosa. Sera of 20 patients with active OP were studied. All sera bound to integrin alpha6 in DU145 cell lysate by immunoprecipitation and immunoblot assay. The same sera bound only to fragment A and its subfragment A2 on an immunoblot assay. The specificity of the binding was further characterized by blocking and cross-absorption studies. A 14-aa synthetic peptide A2.1, within fragment A2, bound to all the test sera. The sera in this study bound to only one epitope. Controls were sera samples from 10 healthy volunteers and 40 patients with other variants of mucous membrane pemphigoid and mAb GoH3 and BQ16 to integrin alpha6. Control sera did not bind to the full-length integrin alpha6 subunit nor any of the cloned fragments. The OP patient sera and immunoaffinity-purified OP sera, rabbit antisera against fragments A and A2, and mAb GoH3 produced basement membrane separation of oral mucosa in organ culture. This study identifies a peptide within the extracellular domain of integrin alpha6 molecule, to which Abs in the sera from patients with OP bind, and which may play an important role in the pathogenesis of OP.