Probing the U-Shaped Conformation of Caveolin-1 in a Bilayer

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive. This work aims to characterize the structure and dynamics of caveolin-1 (D82–S136; Cav1_82–136) in a DMPC bilayer using NMR, fluorescence emission measurements, and molecular dynamics simulations. The secondary structure of Cav1_82–136 from NMR chemical shift indexing analysis serves as a guideline for generating initial structural models. Fifty independent molecular dynamics simulations (100 ns each) are performed to identify its favorable conformation and orientation in the bilayer. A representative configuration was chosen from these multiple simulations and simulated for 1 μs to further explore its stability and dynamics. The results of these simulations mirror those from the tryptophan fluorescence measurements (i.e., Cav1_82–136 insertion depth in the bilayer), corroborate that Cav1_82–136 inserts in the membrane with U-shaped conformations, and show that the angle between H1 and H2 ranges from 35 to 69°, and the tilt angle of Cav1_82–136 is 27 ± 6°. The simulations also reveal that specific faces of H1 and H2 prefer to interact with each other and with lipid molecules, and these interactions stabilize the U-shaped conformation.     

Modulation of the transferrin receptor during DMSO-induced differentiation in HL-60 cells

When HL-60 cells are induced to differentiate by dimethyl sulfoxide along a granulocytic pathway there is a fivefold decrease in the total number of transferrin receptors within 3 days, as compared to untreated cells. This decrease is due primarily to a rapid decline in the synthesis of the receptor rather than an increase in the degradation of the receptor. The decrease in transferrin receptor synthesis is a specific and early event that precedes the cessation of cell proliferation, differentiation, and the decrease in total protein synthesis.     

Quantitation of mitochondrial injury in cultured rat heart endothelioid cells with nitroblue tetrazolium

Synopsis A sensitive method is presented for measurement of changes in the permeability of mitchondria in cultured cells. Rat heart endothelioid cells were used to determine the penetration rate of nitroblue tetrazolium (NitroBT) or other reactants into mitochondriain situ. Nitroblue formazan, produced as a consequence of succinate dehydrogenase activity in the mitochondria, was eluted and measured with a spectrophotometer. Prior injury of cells with hypo-osmolar solutions increased the rate of formazan production. Several methods are described or suggested for the statistical analysis of the data.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 M PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 M PAA) and with a 16-h light/8-h dark photoperiod (500 M PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 M PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 M as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 M PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 M.Paper No. 88514 of the Journal Series of the Idaho Agricultural Experiment Station, Moscow, Idaho, USA.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 μM PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 μM PAA) and with a 16-h light/8-h dark photoperiod (500 μM PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 μM PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 μM as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 μM PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 μM.     

Studies of feedback suppression of bile salt synthesis in the bile-fistula rat

We have previously reported that intravenous infusion of taurocholate at 10 mumol (100 g.hr) into bile-fistula rats suppressed bile salt synthesis by 85% (Pries et al. 1983. J. Lipid Res. 24: 141-146). Recently, however, infusion rates twice this high have been reported not to suppress synthesis (Davis et al. 1984. Falk Symposium 42. MTP Press Ltd., Boston. 37-45). Because the only major difference in design of these two studies was supplementation with sodium bicarbonate to replace biliary losses induced by bile salt choleresis, we have repeated our studies with and without bicarbonate supplementation. Without bicarbonate, as before, we found suppression of synthesis during infusion of taurocholate at 10 mumol/(100 g.hr). With bicarbonate, no suppression of synthesis occurred at these infusion rates. These data indicate that bicarbonate supplementation is essential when testing physiological effects of infused bile salt in the bile-fistula rat.     

Characterization of proteins that associate with an unglycosylated form of the transferrin receptor in A431 cells

A protein doublet (Mr = 135,000/130,000) was found to coprecipitate with an unglycosylated form of the transferrin receptor in tunicamycin-treated A431 cells. This doublet is not detected with either a monoclonal or polyclonal antibody to the transferrin receptor on Western blots indicating that these proteins do not interact directly with transferrin receptor antibody. Proteolytic digestion patterns of the individual proteins of the Mr = 135,000/130,000 doublet suggest that they are related to one another and are distinct from the transferrin receptor. Further characterization of these proteins indicates that they form a high molecular weight complex with the unglycosylated but not the glycosylated form of the transferrin receptor. Pulse-chase experiments demonstrate that the proteins post-translationally associate with the receptor.     

Rhesus monkey (Macaca mulatta) complex learning skills reassessed

Results from three experiments on basic learning and transfer in rhesus monkeys (Macaca mulatta) are reported in which fully automated testing paradigms, afforded by the Language Research Center's Computerized Test System (LRC-CTS), were employed. Performance levels for discrimination learning set, transfer index, and mediational-learning testing were uniformly higher than was predicted from the literature, in contrast to previous reports of compromised learning under similar conditions (automated apparatus, planimetric stimuli, spatial discontiguity between stimuli and response loci). Analyses reveal relatively advanced learning set performance, transfer-index ratios, and positive transfer of learning even at stringent criterion levels. Moreover, the data suggest that rhesus monkeys tested in these experiments exhibit mediational instead of associative learning strategies, as do great apes and in contrast to previous reports of rhesus learning. We argue that the LRC-CTS enhances learning by nonhuman primate subjects, obviating those factors, reported in the literature from experiments in which manual or other automated systems were employed, that compromise learning.     

The accessibility of etheno-nucleotides to collisional quenchers and the nucleotide cleft in G- and F-actin.

Recent publication of the atomic structure of G-actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C., 1990, Nature 347, 37-44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno-nucleotides bound to G- and F-actin were examined in order to assess polymerization-related changes in the nucleotide phosphate site. The Mg(2+)-induced polymerization of actin quenched the fluorescence of the etheno-nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F-actin than G-actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G- and F-actin was greatest for I-, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization-related movements of charged residues in the atomic structure of G-actin are suggested. The binding of S-1 to epsilon-ATP-G-actin increased the accessibility of the etheno group to I- even over that in Mg(2+)-polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S-1 to actin. Thus, the binding of S-1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin.(ABSTRACT TRUNCATED AT 250 WORDS)