The action of defined oxygen-centred free radicals on human low-density lipoprotein.

The effects of defined oxygen-centred free radicals on human low-density lipoprotein (LDL) structure and receptor affinity are discussed in relation to the mechanisms of cell-mediated oxidative modification of LDL. Both hydroxyl (OH.) and hydroperoxyl (HO2.) radicals caused depletion of endogenous alpha-tocopherol and formation of hydroperoxides. Superoxide (O2-.) radicals produced only very limited oxidation, but could potentiate oxidation stimulated by the addition of Cu2+. All these radicals enhanced the net negative charge of intact LDL and induced fragmentation of apolipoprotein B-100 (apo B). OH. also caused cross-linking of apo B. Radical attack decreased the affinity of LDL for the fibroblast apo B/E receptor, but did not enhance its endocytosis by mouse macrophages.     

Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.     

Hybridization between two parapatric ranid frog species in the northern Sierra Nevada,California, USA

Contact zones between species provide a unique opportunity to test whether taxa can hybridize or not. Cross‐breeding or hybridization between closely related taxa can promote gene flow (introgression) between species, adaptation, or even speciation. Though hybridization events may be short‐lived and difficult to detect in the field, genetic data can provide information about the level of introgression between closely related taxa. Hybridization can promote introgression between species, which may be an important evolutionary mechanism for either homogenization (reversing initial divergence between species) or reproductive isolation (potentially leading to speciation). Here, we used thousands of genetic markers from nuclear DNA to detect hybridization between two parapatric frog species (Rana boylii and Rana sierrae) in the Sierra Nevada of California. Based on principal components analysis, admixture, and analysis of heterozygosity at species diagnostic SNPs, we detected two F1 hybrid individuals in the Feather River basin, as well as a weak signal of introgression and gene flow between the frog species compared with frog populations from two other adjacent watersheds. This study provides the first documentation of hybridization and introgression between these two species, which are of conservation concern.     

Hexose phosphorylation and the putative calcium channel component Mid1p are required for the hexose-induced transient elevation of cytosolic calcium response in Saccharomyces cerevisiae

Saccharomyces cerevisiae responds to environ-mental stimuli such as an exposure to pheromone or to hexoses after carbon source limitation with a transient elevation of cytosolic calcium (TECC) response. In this study, we examined whether hexose transport and phosphorylation are necessary for the TECC response. We found that a mutant strain lacking most of the known hexose transporters was unable to carry out the TECC response when exposed to glucose. A mutant strain that lacked the ability to phosphorylate glucose was unable to respond to glucose addition, but displayed a normal TECC response after the addition of galactose. These results indicate that hexose uptake and phosphorylation are required to trigger the hexose-induced TECC response. We also found that the TECC response was significantly smaller than normal when the level of environmental calcium was reduced, and was abolished in a mid1 mutant that lacked a subunit of the high-affinity calcium channel of the yeast plasma membrane. These results indicate that most or all of the TECC response is mediated by an influx of calcium from the extracellular space. Our results indicate that this transient increase in plasma membrane calcium permeability may be linked to the accumulation of Glc-1-P (or a related glucose metabolite) in yeast.     

A Saccharomyces cerevisiae mutant unable to convert glucose to glucose-6-phosphate accumulates excessive glucose in the endoplasmic reticulum due to core oligosaccharide trimming

D-Glucose is the preferred carbon and energy source for most eukaryotic cells. Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P). The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P. In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose. However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal. Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu(3)Man(9)GlcNac(2) effectively reduced the accumulation of glucose. Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5Delta or alg6Delta, also diminished glucose accumulation. These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER). Consistent with this conclusion, both [(14)C]glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P. The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway. Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca(2+) uptake. This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation.     

Incidence and levels of fumonisin contamination in Colombian corn and corn products

A survey was conducted to determine the levels of fumonisins B1 and B2 in corn and corn-based products available in Colombia for human and animal consumption. A total of 120 samples were analyzed by acetonitrile-water extraction, cleanup with a strong-anion-exchange column, and liquid chromatography with o-phthaldialdehyde-2-mercaptoethanol derivatization and fluorescence detection. The samples of corn and corn-based products for animal intake were taken at different feed manufacturing plants, whereas the samples used for human foods where purchased from local retail stores. The number of positive samples for fumonisin B1 was 20.0% higher in corn and corn-based products for animal intake (75.0%) than in corn and corn-based products for human consumption (55.0%). The levels of fumonisin B1 were also higher in corn and corn-based products for animal intake (mean = 694 μg/kg; range = 32–2964 μg/kg), than in corn and corn-based products for human intake (mean = 218 μg/kg; range = 24–2170 μg/ kg). The incidence and levels of fumonisin B2 were lower than those for fumonisin B1. Corn and corn-based products for animal consumption had an incidence of fumonisin B2 of 58.3%, with a mean value of 283 μg/kg, and a range of 44–987 μg/kg. The incidence of fumonisin B2 in corn-based products for human intake was 35.0%, with a mean value of 118 μg/kg and a range of 21–833 μg/kg. The highest incidence and levels of fumonisins were found in samples of hominy feed, with concentrations ranging from 86 to 2964 μg/kg fumonisin B1 and 57 to 987 μg/kg fumonisin B2.     

Enhancement of alveolar epithelial sodium channel activity with decreased cystic fibrosis transmembrane conductance regulator expression in mouse lung

We sought to establish whether the cystic fibrosis transmembrane conductance regulator (CFTR) regulates the activity of amiloride-sensitive sodium channels (ENaC) in alveolar epithelial cells of wild-type, heterozygous (Cftr(+/-)), knockout (Cftr(-/-)), and ΔF508-expressing mice in situ. RT-PCR studies confirmed the presence of CFTR message in freshly isolated alveolar type II (ATII) cells from wild-type mice. We patched alveolar type I (ATI) and ATII cells in freshly prepared lung slices from these mice and demonstrated the presence of 4-pS ENaC channels with the following basal open probabilities (P(o)): wild-type=0.21 ± 0.015: Cftr(+/-)=0.4 ± 0.03; ΔF508=0.55 ± 0.01; and Cftr(-/-)=and 0.81 ± 0.016 (means ± SE; n ≥ 9). Forskolin (5 μM) or trypsin (2 μM), applied in the pipette solution, increased the P(o) and number of channels in ATII cells of wild-type, Cftr(+/-), and ΔF508, but not in Cftr(-/-) mice, suggesting that the latter were maximally activated. Western blot analysis showed that lungs of all groups of mice had similar levels of α-ENaC; however, lungs of Cftr(+/-) and Cftr(-/-) mice had significantly higher levels of an α-ENaC proteolytic fragment (65 kDa) that is associated with active ENaC channels. Our results indicate that ENaC activity is inversely correlated to predicted CFTR levels and that CFTR heterozygous and homozygous mice have higher levels of proteolytically processed ENaC fragments in their lungs. This is the first demonstration of functional ENaC-CFTR interactions in alveolar epithelial cells in situ.     

Inhibition of phosphoglucomutase activity by lithium alters cellular calcium homeostasis and signaling in Saccharomyces cerevisiae

Phosphoglucomutase is a key enzyme of glucose metabolism that interconverts glucose-1-phosphate and glucose-6-phosphate. Loss of the major isoform of phosphoglucomutase in Saccharomyces cerevisiae results in a significant increase in the cellular glucose-1-phosphate-to-glucose-6-phosphate ratio when cells are grown in medium containing galactose as carbon source. This imbalance in glucose metabolites was recently shown to also cause a six- to ninefold increase in cellular Ca²⁺ accumulation. We found that Li⁺ inhibition of phosphoglucomutase causes a similar elevation of total cellular Ca²⁺ and an increase in ⁴⁵Ca²⁺ uptake in a wild-type yeast strain grown in medium containing galactose, but not glucose, as sole carbon source. Li⁺ treatment also reduced the transient elevation of cytosolic Ca²⁺ response that is triggered by exposure to external CaCl₂ or by the addition of galactose to yeast cells starved of a carbon source. Finally, we found that the Ca²⁺ overaccumulation induced by Li⁺ exposure was significantly reduced in a strain lacking the vacuolar Ca²⁺-ATPase Pmc1p. These observations suggest that Li⁺ inhibition of phosphoglucomutase results in an increased glucose-1-phosphate-to-glucose-6-phosphate ratio, which results in an accelerated rate of vacuolar Ca²⁺ uptake via the Ca²⁺-ATPase Pmc1p. calcium influx; calcium signal; galactose; glucose phosphate