Expression of caveolin-1 enhances cholesterol efflux in hepatic cells

HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains (caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane.     

Grassland yield declined by a quarter in 5 years of free-air ozone fumigation

The sensitivity of seminatural grasslands to ozone (O₃) pollution is not well known, in spite of the important function of these common ecosystems for agriculture and nature conservation. A 5‐year field experiment was carried out at a rural, mid‐elevation site at Le Mouret (Switzerland) to investigate the effect of elevated O₃ on yield and species composition of a permanent, extensively managed, species‐rich old pasture. Using a free‐air fumigation system, circular plots of 7 m diameter were exposed to either ambient air (control plots) or to air containing O₃ of approximately 1.5 × ambient concentrations. The resulting accumulated O₃ exposure over the threshold of 40 ppb for one season ranged from 13.3 to 59.5 ppm h in the elevated O₃ plots, and from 1.0 to 20.7 ppm h in the control plots. Subplots in each ring were harvested three times each year, and harvested biomass was separated into functional groups (FGs) (grasses, legumes, forbs). Productivity in both treatments decreased over time, but the yield of O₃‐exposed plots decreased faster than that of the control plots, with the reduction being twice as large by the end of the fifth season. Compared with the ambient air control, loss in annual dry matter yield was 23% after 5 years. The change in annual biomass production because of O₃ stress was accompanied by a change in the fractions of FGs, with the legume fraction showing a strong negative response. Such long‐term effects of O₃ stress could have negative implications for the maintenance of biological diversity in rural landscapes across large areas of Europe. The results from this first long‐term experiment show that a moderately elevated O₃ level reduces the productivity of intact grasslands during a 5‐year exposure under real field conditions.     

Detection of Enterobacter sakazakii in Dried Infant Milk Formula by Cationic-Magnetic-Bead Capture

Enterobacter sakazakii has been associated with life-threatening infections in premature low-birth-weight infants. Contaminated infant milk formula (IMF) has been implicated in cases of E. sakazakii meningitis. Quick and sensitive methods to detect low-level contamination sporadically present in IMF preparations would positively contribute towards risk reduction across the infant formula food chain. Here we report on the development of a simple method, combining charged separation and growth on selective agar, to detect E. sakazakii in IMF. This protocol can reliably detect 1 to 5 CFU of E. sakazakii in 500 g of IMF in less than 24 h.     

A missense mutation in AGTPBP1 was identified in sheep with a lower motor neuron disease

A type of lower motor neuron (LMN) disease inherited as autosomal recessive in Romney sheep was characterized with normal appearance at birth, but with progressive weakness and tetraparesis after the first week of life. Here, we carried out genome-wide homozygosity mapping using Illumina Ovine SNP50 BeadChips on lambs descended from one carrier ram, including 19 sheep diagnosed as affected and 11 of their parents that were therefore known carriers. A homozygous region of 136 consecutive single-nucleotide polymorphism (SNP) loci on chromosome 2 was common to all affected sheep and it was the basis for searching for the positional candidate genes. Other homozygous regions shared by all affected sheep spanned eight or fewer SNP loci. The 136-SNP region contained the sheep ATP/GTP-binding protein 1 (AGTPBP1) gene. Mutations in this gene have been shown to be related to Purkinje cell degeneration (pcd) phenotypes including ataxia in mice. One missense mutation c.2909G>C on exon 21 of AGTPBP1 was discovered, which induces an Arg to Pro substitution (p.Arg970Pro) at amino-acid 970, a conserved residue for the catalytic activity of AGTPBP1. Genotyping of this mutation showed 100% concordant rate with the recessive pattern of inheritance in affected, carrier, phenotypically normal and unrelated normal individuals. This is the first report showing a mutant AGTPBP1 is associated with a LMN disease in a large mammal animal model. Our finding raises the possibility of human patients with the same etiology caused by this gene or other genes in the same pathway of neuronal development.     

EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization

Eps15 homology domain-containing 2 (EHD2) belongs to the EHD-containing protein family of dynamin-related ATPases involved in membrane remodeling in the endosomal system. EHD2 dimers oligomerize into rings on highly curved membranes, resulting in stimulation of the intrinsic ATPase activity. In this paper, we report that EHD2 is specifically and stably associated with caveolae at the plasma membrane and not involved in clathrin-mediated endocytosis or endosomal recycling, as previously suggested. EHD2 interacts with pacsin2 and cavin1, and ordered membrane assembly of EHD2 is dependent on cavin1 and caveolar integrity. While the EHD of EHD2 is dispensable for targeting, we identified a loop in the nucleotide-binding domain that, together with ATP binding, is required for caveolar localization. EHD2 was not essential for the formation or shaping of caveolae, but high levels of EHD2 caused distortion and loss of endogenous caveolae. Assembly of EHD2 stabilized and constrained caveolae to the plasma membrane to control turnover, and depletion of EHD2, resulting in endocytic and more dynamic and short-lived caveolae. Thus, following the identification of caveolin and cavins, EHD2 constitutes a third structural component of caveolae involved in controlling the stability and turnover of this organelle.     

Interactions of rab5 with cytosolic proteins.

Rab proteins, one of the subfamilies of ras-like small GTP-binding proteins, are attached to cellular compartments or transport vesicles and may determine the specificity of fusion between these compartments and vesicles. It has been proposed that they alternate between a membrane-bound and a cytosolic state during their functional cycle. We have used a photo-crosslinking approach to identify their cytosolic interaction partners. In vitro synthesized rab5 was cross-linked in the presence of ATP mainly to three cytosolic proteins of 52, 65, and 85 kDa. Sucrose density gradient centrifugation of the cross-linked products suggested that they were part of a 10-14 S complex. Furthermore, rab5 was cross-linked to these and additional cytosolic proteins of 42, 48, and 160 kDa in the absence of ATP. Unexpectedly, upon ATP depletion of the cytosol cross-linked and noncross-linked rab5 was found in a sedimentable high molecular weight structure. Other members of the rab subfamily, but not N-ras, also sedimented under these conditions. Electrophoretic and electron microscopic analysis of the pelleted material revealed that it contained actin filament bundles and intermediate filaments. Our data suggest that cytosolic rab proteins interact with several proteins in a 10-14 S complex, and that the rab proteins may interact directly or indirectly via this complex with the cytoskeleton.     

Membrane microdomains and caveolae.

Glycosphingolipid- and cholesterol-enriched microdomains, or rafts, within the plasma membrane of eukaryotic cells have been implicated in many important cellular processes, such as polarized sorting of apical membrane proteins in epithelial cells and signal transduction. Until recently, however, the existence of such domains remained controversial. The past year has brought compelling evidence that microdomains indeed exist in living cells. In addition, several recent papers have suggested that caveolae, which are considered to be a specific form of raft, and caveolins, the major membrane proteins of caveolae, are involved in the dynamic cholesterol-dependent regulation of specific signal transduction pathways.     

Effects of glucose infusion on surfactant and glycogen regulation in fetal lamb lung

To investigate the increased incidence of respiratory distress syndrome (RDS) that occurs in infants of diabetic mothers (IDM) with poor maternal glucose homeostasis, we infused glucose intravenously at a rate of 14 +/- 2 (SD) mg.kg-1.min-1 into eight twin and four singleton chronically catheterized fetal lambs from 112 days (0.77) gestation onward. Twelve catheterized and seven uncatheterized fetuses served as controls, including the eight twins of the glucose-treated fetuses. Glucose infusion resulted in a twofold elevation in fetal serum glucose levels and a 2.2-fold elevation in fetal serum insulin levels. Before 113 days (0.9) gestation, pulmonary disaturated phosphatidylcholine (DSPC) content was 1.5-fold higher in the glucose-infused fetuses than in the controls. However, after 0.9 gestation, pulmonary DSPC content increased 2.2-fold in the controls but did not increase significantly in the glucose-infused fetuses. In addition, the DSPC content of lung lavage was 5.0-fold higher in the controls and lung stability to air inflation was 2.0-fold greater and to deflation was 2.2-fold greater than in the glucose-infused fetuses. Pulmonary adenosine 3',5'-cyclic monophosphate-dependent protein kinase activity was also 1.5-fold higher, and pulmonary protein kinase C activity was 1.3-fold higher in the controls than in the glucose-infused fetuses. In contrast, glucose infusion was associated with a 1.8-fold increase in pulmonary glycogen content and with increased activities of glycogen phosphorylase kinase and glycogen phosphorylase. We conclude that the effects of chronic glucose infusion on fetal lamb lung DSPC and lung stability are compatible with a predisposition of the fetus to develop RDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenylate cyclase activity during phenotypic variation of Bordetella pertussis

During MgSO4-induced modulation of Bordetella pertussis, adenylate cyclase activity, histamine-sensitizing activity (HSA) and the major cell-envelope polypeptides with Mr 28000 and 30000 (X polypeptides) were lost synchronously at a rate which could be accounted for by a simple growth-dilution effect. MgSO4 and other compounds which induced the above phenotypic change caused little inhibition of adenylate cyclase activity. Nicotinic acid was the sole exception and at 4.1 mM-caused 60% inhibition of activity. Lysates of modulated cells, mixed with lysates of unmodulated cells, had no effect on either adenylate cyclase activity or HSA. Protein synthesis was a prerequisite for MgSO4-induced modulation and also for the reversal of this process. Exogenous cAMP and dibutyryl cAMP (5 mM) had no counteracting effect on MgSO4- or nicotinic acid-induced modulation. The concentration of MgSO4 required to induce loss of the X polypeptides (10 to 11 mM) was not altered by promoting adenylate cyclase activity by including an activator in the growth medium. In one culture containing 10 mM-MgSO4 and activator, partial loss of the X polypeptides occurred and yet the extracellular cAMP concentration was twice that of cultures without activator and where full expression of the X polypeptides occurred. [3H]cAMP-binding activity was detected in cell extracts of several strains of B. pertussis, but antiserum against purified Escherichia coli catabolite repressor protein gave no reaction with B. pertussis cell extracts. Respiration rates with amino acids were similar for modulated and unmodulated variants and an avirulent strain of B. pertussis. These results are discussed in relation to a possible causal role for adenylate cyclase in modulation of B. pertussis.