Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

Cytoplasmic dynein nomenclature

A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.     

Somatostatin and insulin release from isolated rat pancreatic islets in response to D-glucose, l-leucine, alpha-ketoisocaproic acid or D-glyceraldehyde: evidence for a regulatory role of adenosine-3' ,5'-cyclic monophosphate.

Somatostatin and insulin release from isolated rat pancreatic islets was stimulated by glucose, leucine or α-ketoisocaproic acid. D-glyceraldehyde stimulated insulin release but diminished the secretion of somatostatin. Glucagon and theophylline amplified the glucose-induced somatostatin release.A regulatory role of the D-cell's adenylate cyclase/phosphodiesterase system for the release of somatostatin is suggested. Furthermore, stimulation as well as inhibition of somatostatin release might be of significance for the secretory function of the B-cell.     

Identification of a microtubule-based cytoplasmic motor in the nematode C. elegans

C. elegans contains a microtubule binding protein that resembles both dynein and kinesin. This protein has a MgATPase activity and copurifies on both sucrose gradients and DEAE Sephadex columns with a polypeptide of Mr approximately 400 kd. The ATPase activity is 50% inhibited by 10 microM vanadate, 1 mM N-ethyl maleimide, or 5 mM AMP-PNP; it is enhanced 50% by 0.2% Triton. The 400 kd polypeptide is cleaved at a single site by ultraviolet light in the presence of ATP and vanadate. In these ways, the protein resembles dynein. The protein also promotes ATP-dependent translocation of microtubules or axonemes, "plus" ends trailing. This property is kinesin-like; however, the motility is blocked by 5 microM vanadate, 1 mM N-ethyl maleimide, 0.5 mM ATP-gamma-S, or by ATP-vanadate-UV cleavage of the 400 kd polypeptide, characteristics that differ from kinesin. We propose that this protein is a novel microtubule translocator.     

Alpha-conotoxins ImI and ImII. Similar alpha 7 nicotinic receptor antagonists act at different sites

A novel conotoxin, alpha-conotoxin ImII (alpha-CTx ImII), identified from Conus imperialis venom ducts, was chemically synthesized. A previously characterized C. imperialis conotoxin, alpha-conotoxin ImI (alpha-CTx ImI), is closely related; 9 of 12 amino acids are identical. Both alpha-CTx ImII and alpha-CTx ImI functionally inhibit heterologously expressed rat alpha7 nAChRs with similar IC(50) values. Furthermore, the biological activities of intracranially applied alpha-CTx ImI and alpha-CTx ImII are similar over the same dosage range, and are consistent with alpha7 nAChR inhibition. However, unlike alpha-CTx ImI, alpha-CTx ImII was not able to block the binding of alpha-bungarotoxin to alpha7 nAChRs. alpha-Conotoxin ImI and alpha-bungarotoxin-binding sites have been well characterized as overlapping and located at the cleft between adjacent nAChR subunits. Because alpha-CTx ImI and alpha-CTx ImII share extensive sequence homology, the inability of alpha-CTx ImII to compete with alpha-BgTx is surprising. Furthermore, functional studies in oocytes indicate that there is no overlap between functional binding sites of alpha-CTx ImI and alpha-CTx ImII. Like alpha-CTx ImI, the block by alpha-CTx ImII is voltage-independent. Thus, alpha-CTx ImII represents a probe for a novel antagonist binding site, or microsite, on the alpha7 nAChR.     

Tightly bound calcium of adenosine triphosphatase in sarcoplasmic reticulum from rabbit skeletal muscle

Sarcoplasmic reticulum vesicles were shown to possess a class of tightly bound calcium ions, inaccessible to the chelator, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid at 0 degrees C or 25 degrees C, amounting to 4.5 nmol/mg of protein (approximately 0.5 mol/mol (Ca2+,Mg2+)-ATPase). The calcium ionophores, A23187 and X537A, induced rapid exchange of tightly bound calcium in the presence of chelator. Chelator alone at 37 degrees C, caused irreversible loss of bound calcium, which correlated with uncoupling of transport from (Ca2+,Mg2+)-ATPase activity. Uncoupling was not accompanied by increased permeability to [14C]inulin. Slow exchange of tightly bound calcium with medium calcium was unaffected by turnover of the ATPase or by tryptic cleavage into 55,000- and 45,000-dalton fragments. Binding studies with labeled calcium suggested that tight binding involves a two-step process: Ca2+ + E in equilibrium K E . Ca2+ leads to E < Ca2+ where E and < Ca2+ represent the ATPase and tightly bound calcium, and K = 1.6 X 10(3) M-1. It is suggested that tightly bound calcium is located in a hydrophobic pocket in, or in close proximity to the ATPase, and, together with tightly bound adenine nucleotides (Aderem, A., McIntosh, D. B., and Berman, M. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3622-03632), is related to the ability of the ATPase to couple hydrolysis of ATP to vectorial transfer of calcium across the membrane.     

Interactions between fish,grazing invertebrates and algae in a New Zealand stream: a trophic cascade mediated by fish-induced changes to grazer behaviour?

Experiments in laboratory stream channels compared the behaviour of Deleatidium mayfly nymphs in the absence of fish with that in the presence of either native common river galaxias (Galaxias vulgaris Stokell) or introduced brown trout (Salmo trutta L.). Galaxias present similar predation risks to prey during day and night but are more active at night. Whereas, trout present a higher predation risk during the day. Deleatidium maintained a fixed nocturnal drift periodicity that is characteristic of streams containing visually feeding fish regardless of the nature of the predation regime presented in the laboratory. However, the number on the substratum surface, and therefore able to graze algae, was lower when fish were present than when they were absent. The number was lower during the day in the presence of trout, when they present the highest predation risk, and lower during the night compared to the day in trials with galaxias when galaxias activity disturbs Deleatidium from the substratum. Increases in the probability of Deleatidium leaving a patch, reductions in the proportion of mayflies on high quality patches and reductions in the distance travelled from refuge also reflected variations in the predation regime. Similar differences in positioning were observed under the same predation regimes in in situ channels in the Shag River and these were associated with differences in algal biomass. Algal ash-free dry mass (AFDM) and chlorophyll a (chl a) were higher on the tops of cobbles when fish were present. Fish also affected the biomass and the distribution of algae on cobbles as AFDM and chl a were higher on the sides of cobbles from channels with trout compared to those with galaxias. Changes in grazing behaviour, caused by predator avoidance, are likely to have been responsible for differences in algal biomass because no significant differences were detected between treatments in the biomass of Deleatidium or of total invertebrates.     

Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization

Background

Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization.

Methods

In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep.

Results

The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation.

Conclusion

Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.