Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.     

Generation and characterization of two novel mouse models exhibiting the phenotypes of the metabolic syndrome: Apob48-/-Lepob/ob mice devoid of ApoE or Ldlr

The metabolic syndrome is a group of disorders including obesity, insulin resistance, atherogenic dyslipidemia, hyperglycemia, and hypertension. To date, few animal models have been described to recapitulate the phenotypes of the syndrome. In this study, we generated and characterized two lines of triple-knockout mice that are deficient in either apolipoprotein E (Apoe(-/-)) or low-density lipoprotein receptor (Ldlr(-/-)) and express no leptin (Lep(ob/ob)) or apolipoprotein B-48 but exclusively apolipoprotein B-100 (Apob(100/100)). These two lines are referred to as Apoe triple-knockout-Apoe 3KO (Apoe(-/-)Apob(100/100)Lep(ob/ob)) and Ldlr triple-knockout-Ldlr 3KO (Ldlr(-/-)Apob(100/100)Lep(ob/ob)) mice. Both lines develop obesity, hyperinsulinemia, hyperlipidemia, hypertension, and atherosclerosis. However, only Apoe 3KO mice are hyperglycemic and glucose intolerant and are more obese than Ldlr 3KO mice. To evaluate the utility of these lines as pharmacological models, we treated both with leptin and found that leptin therapy ameliorated most metabolic derangements. Leptin was more effective in improving glucose tolerance in Ldlr 3KO than Apoe 3KO animals. The reduction of plasma cholesterol by leptin in Ldlr 3KO mice can be accounted for by its suppressive effect on food intake. However, in Apoe 3KO mice, leptin further reduced plasma cholesterol independently of its effect on food intake, and this improvement correlated with a smaller plaque lesion area. These effects suggest a direct role of leptin in modulating VLDL levels and, likewise, the lesion areas in VLDL-enriched animals. These two lines of mice represent new models with features of the metabolic syndrome and will be useful in testing therapies targeted for combating the human condition.     

Analysis of binding sites and efficacy of a species-specific peptide at rat and human neurotensin receptors.

We have developed a neurotensin analog, L-[3,1'-naphthylalanine11]NT(8-13), NT34, that can distinguish between rat and human neurotensin receptors, and exhibits more than a 100-fold difference in binding affinities and a 60-fold difference in functional coupling to phosphatidylinositol turnover. Using cells transfected with different numbers of the appropriate receptors, we measured the changes in phosphatidylinositol production, and then evaluated the efficiency of receptor-effector coupling based on Furchgott's design. The binding of NT34 at both rat and human neurotensin receptors stably expressed in CHO-K1 cells was to two sites, while the binding of NT was to one site. At the rat receptor the equilibrium dissociation constant (Kd) for NT34 at the high-affinity site was 0.058 nM, while that at the low-affinity site was 3.1 nM. For the human receptor at the high-affinity site, the Kd for NT34 was 18 nM, while that at the low-affinity site was 180 nM. For both species the percentage of receptors representing the high-affinity site was approximately 60-70% with 30-40% at the low-affinity site. We derived agonist dissociation constants (Ka) for NT and NT34, which suggest that for NT34, the low-affinity site is functionally coupled to phosphatidylinositol turnover. Finally, we compared the relative efficacies of both compounds and found that NT34 was about 2-fold and 4-fold more efficacious than NT in stimulating phosphatidylinositol turnover in rat and human NT receptors, respectively.     

Intrachromosomal homologous recombination in human cells which differ in nucleotide excision-repair capacity

To examine the mechanism of recombination and the role of DNA repair in this process, we transfected a plasmid carrying duplicated copies of the Herpes simplex virus I thymidine kinase (Htk) gene, each containing an 8 bp XhoI site inserted in a unique site and with the neo coding for geneticin resistance located between them, into tk-deficient human cell lines which differ in their ability to carry out nucleotide excision repair. One parental cell line has a normal level of repair activity; the second has an intermediate level, and the third has virtually no repair activity. Several geneticin-resistant transfectant cell strains from each parental line were isolated and assayed for the ability to undergo productive recombination giving rise to tk+ cells. Approximately 25% of them could do so. Southern blot analysis of these transfectants indicated that the majority contained a single copy, or at most, two copies of the plasmid integrated into the chromosome. Fluctuation analysis tests to determine the rate of spontaneous recombination (events per 10(6) cells per cell generation) in the various cell strains showed that the rates ranged from 0.15 to 4.1. The mean rate for the cell strains derived from the repair-deficient cell line was 3.6; for those derived from the cells with an intermediate rate, it was 0.8; and for those with a normal rate of excision repair, it was 0.9. Southern blot analysis of tk+ recombinants showed that in all cases, one of the Htk genes had become wild-type, i.e., XhoI-resistant. 90% of the recombinants retained the Htk gene duplication, consistent with non-reciprocal transfer of genetic information, i.e., gene conversion. The rest contained a single, wild-type Htk gene, consistent with a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. These cell strains will be useful for investigating the role of DNA damage and repair in homologous recombination.     

Analysis of the Factors Involved in the Retinoic Acid-Induced Differentiation of a Retinoid-Hypersensitive Embryonal Carcinoma Cell Mutant

Retinoic acid (RA) has been known to play an important role in cellular growth and differentiation as well as in vertebrate development. Many in vitro cell cultures also respond to RA by differentiating. Perhaps the most widely studied of these cultures are embryonal carcinoma (EC) cells. We have used an RA-hypersensitive EC cell mutant, created by retroviral insertion, to analyze the activity of the identifiable components in the RA response pathway. We have analyzed the mRNA expression patterns of the retinoic acid receptors (RARs) α, β, and γ, the retinoid X receptors (RXRs) α, β, and γ, and the cellular retinoic acid binding proteins (CRABPs) I and II. Our results indicate that CRABP I, RAR β, and RAR γ mRNAs are expressed differentially between parent and RA-hypersensitive mutant cells. All three messages are present at higher basal levels and at earlier times after RA addition in the mutant relative to parental cells. All other elements examined are equivalently expressed. Therefore analyses of the expression patterns of CRABPs, RARs, and RXRs in these RA-hypersensitive cells point to the probable importance of CRABP I, RAR β, and RAR γ in the RA induction pathway and also indicate that CRABP II and RXR γ are not likely to be critical elements in the early differentiative response of cells to RA.     

Direct and size-mediated effects of temperature and ration-dependent growth rates on energy reserves in juvenile anadromous alewives (Alosa pseudoharengus)

Growth rate and energy reserves are important determinants of fitness and are governed by endogenous and exogenous factors. Thus, examining the influence of individual and multiple stressors on growth and energy reserves can help estimate population health under current and future conditions. In young anadromous fishes, freshwater habitat quality determines physiological state and fitness of juveniles emigrating to marine habitats. In this study, the authors tested how temperature and food availability affect survival, growth and energy reserves in juvenile anadromous alewives (Alosa pseudoharengus), a forage fish distributed along the eastern North American continent. Field-collected juvenile anadromous A. pseudoharengus were exposed for 21 days to one of two temperatures (21°C and 25°C) and one of two levels of food rations (1% or 2% tank biomass daily) and compared for differences in final size, fat mass-at-length, lean mass-at-length and energy density. Increased temperature and reduced ration both led to lower growth rates, and the effect of reduced ration was greater at higher temperature. Fat mass-at-length decreased with dry mass, and energy density increased with total length, suggesting size-based endogenous influences on energy reserves. Lower ration also directly decreased fat mass-at-length, lean mass-at-length and energy density. Given the fitness implications of size and energy reserves, temperature and food availability should be considered important indicators of nursery habitat quality and incorporated in A. pseudoharengus life-history models to improve forecasting of population health under climate change.