Thermotropic lipid phase separation in the human immunodeficiency virus

The presence of thermodependent lipid domains in the envelope of the human immunodeficiency virus (HIV) was studied. HIV was propagated in Hut-78 cells and purified by differential-gradient centrifugation. Since the virus was highly infectious in cell culture and Western blots of detergent-inactivated HIV showed envelope proteins when exposed to sera containing anti-HIV antibodies, this viral preparation was not deficient in 'spike' or 'knob' particles. Electron spin resonance (ESR) studies of intact HIV labeled with 5-nitroxide stearate (5-NS) indicated that a temperature-dependent lipid phase separation occurs with a high onset at approx. 42 degrees C and a low onset at approx. 15 degrees C. Cooling below 42 degrees C induces 5-NS clustering. Similar phase separations with high onsets at approx. 37-38 degrees C were previously identified in 5-NS labeled human erythrocytes (cholesterol/phospholipid (C/P) molar ratio = 0.90) and cholesterol-loaded (C/P = 0.85-0.98) rat liver plasma membranes. These were attributed to a temperature-sensitive redistribution of endogenous lipid components such that 5-NS is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains at low temperatures. Since HIV has a lipid envelope with a similarly high C/P of 0.88 (Aloia et al. (1988) Proc. Natl. Acad. Sci. USA 85, 900-904), cholesterol-rich and cholesterol-poor domains also probably exist in HIV at physiologic temperatures. The reduced stability and infectivity of HIV noted on heating above 42 degrees C may be due, in part, to the abolition of these thermodependent domains.     

The structure of crystalline Escherichia coli-derived rat intestinal fatty acid-binding protein at 2.5-A resolution

Rat intestinal fatty acid-binding protein (I-FABP) is an abundant cytoplasmic protein which is synthesized in the small intestinal lining cell where it is thought to participate in the absorption and intracellular metabolism of fatty acids. Each mole of this 132-residue polypeptide binds 1 mol of long chain fatty acid in a noncovalent fashion. Because of its small size and single ligand-binding site, I-FABP represents an attractive model for defining the molecular details of long chain fatty acid-protein interactions. The structure of Escherichia coli-derived rat I-FABP has now been solved to 2.5 A resolution using three isomorphous heavy atom derivatives. The protein consists of 10 anti-parallel beta-strands present as two orthogonal beta-sheets. Together a "clam shell-like" structure is formed with an opening located between two beta-strands and an interior that is lined with the side chains of nonpolar amino acids. The bound fatty acid ligand is located in the interior of the protein and has a bent conformation, possibly reflecting the presence of several gauche bonds in the hydrocarbon tail. Our present interpretation of the electron density map suggests that the fatty acid is oriented with its carboxylate group facing the guanidinium group of Arg127, whereas the end of its hydrocarbon tail is in close proximity to Val106. The indole side chain of Trp83 forms the molecular framework around which the principal bend of the hydrocarbon chain occurs.     

Nonlinear stiffness--force relationships in whole mammalian skeletal muscles

Small, sinusoidal length changes were superimposed on isometric contractions of fast- and slow-twitch mouse muscles, which were stimulated maximally via their nerves. Stiffness increased with increasing frequency of sinusoidal stimulation, but the relative time course of force and stiffness changes during twitch, tetanic, or partially fused contractions was quite invariant over a range of frequencies in both muscles. Typically, stiffness increases more rapidly than force during contraction and decreases less rapidly during relaxation. This pattern was observed at various temperatures and with various numbers of stimuli. It can be described by a nonlinear relation between stiffness and force with some hysteresis. The presence in the muscle of parallel and series elastic elements, whose stiffness varies with force, may account for the nonlinear relation. This nonlinearity can be used to relate the patterns for summation of force and stiffness observed with brief trains of stimuli under a variety of conditions.     

Cytogenetics of human sperm: meiotic segregation in two translocation carriers.

Meiotic segregation products were studied in sperm from two men heterozygous for the reciprocal translocations t(8;15)(p22;q21) and t(3;16)(p23;q24). A total of 226 and 201 sperm complements, respectively, were analyzed. In each translocation, 63% of complements were unbalanced, and alternate and adjacent 1 percentages were similar. The 3:1 segregation frequencies produced by the two translocations were 3.5% and 5.0%.     

Isolation and partial characterization of endothelial cell extracellular complexes

Human endothelial cells release components into the growth medium that stimulate cell-substratum adhesion. Several macromolecular components were isolated by ultracentrifugation of the endothelial cell conditioned medium. The components were heterogeneous, consisting of several sizes when examined by sedimentation velocity and gel filtration. When the extracellular components were evaluated by electron microscopy, structurally discrete particles were observed. The extracellular components and the complexes mediated cell-substratum adhesion to both human umbilical and arterial endothelial cells. The majority of the extracellular components that promote endothelial cell adhesion were pelleted by ultracentrifugation. Although the complexes contained fibronectin, antibodies to fibronectin did not inhibit cell adhesion to the complexes. Significant inhibition of endothelial cell adhesion was observed in the presence of heparin and heparan sulfate. The supernatant fraction following ultracentrifugation of the growth medium contained a component that suppressed endothelial cell adhesion to culture dishes coated with fibronectin, type I collagen, and endothelial cell complexes. SDS-polyacrylamide gel electrophoresis indicated that the complexes contained several components, and the majority of the large-molecular-weight components were pelleted by ultracentrifugation. The conditioned medium from human endothelial cells contains specific complexes that promote cell-substratum adhesion and components that suppress cell-substratum adhesion.     

Assisted fertilization by zona drilling: a mouse model for correction of oligospermia

A micromanipulation apparatus was used to produce holes in the zonae pellucidae of unfertilized mouse oocytes. A microneedle loaded with acid Tyrode's solution was brought into contact with the zona surface, and positive flow was used in conjunction with mechanical pressure to cause a localized dissolution of the zona. Treated eggs were then fertilized in vitro in comparison with control cells. The zona drilling procedure decreased the sperm count required to achieve fertilization by a factor of approximately 100. The rate of polyspermy in zona-drilled oocytes was not greater than in controls, and oocytes fertilized after drilling, when implanted into pseudopregnant foster females, developed to term at the same rate as controls. The results demonstrate that zona drilling is a safe, effective method of increasing the efficiency of fertilization in vitro and may be useful both in agriculture and medicine for conferring fertility upon males with low sperm counts.     

Differentiation among populations of Sedum wrightii (Crassulaceae) in response to limited water availability: water relations,CO2 assimilation,growth and survivorship

Summary Sedum wrightii is one of only a few species in the Crassulaceae for which there is evidence for a high degree of variability in the ratio of daytime to nighttime CO₂ assimilation. There are both environmental and genetic components to this variability. S. wrightii grows over a wide altitudinal gradient. The purpose of this study was to compare low, intermediate, and high altitude populations with respect to the degree of CAM expression and the capability to tolerate limited water availability. We utilized clonallyreplicated genotypes of plants from each population in common environment greenhouse experiments. Genetic differences among the populations were found in long-term water use efficiency, in 24 hour CO₂ exchange patterns, in biomass ¹³C values, in carbon allocation, and in water status and ultimately survival during prolonged drought. The differences among the populations appear to be closely related to differences in the native habitats. The low altitude, desert plants had the greatest ability to grow and survive under conditions of limited water availability and appear to have the greatest shift to nighttime CO₂ uptake during periods without water, while the high altitude plants had the poorest performance under these conditions and appear to shut down net carbon uptake when severely water limited.     

The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface

The time course of the extracellular reaction sequence ATP----ADP----AMP----adenosine has been examined during recirculation of substrate solutions over cultured pig aortic endothelial cells attached to polystyrene beads. This permits the study of reactions at volume to cell surface ratios approaching those of small blood vessels. When endothelial cells were presented with an initial bolus of ATP, high concentrations of the intermediates ADP and AMP developed before significant conversion of AMP to adenosine occurred. Further, the higher the initial ATP concentration, the slower the conversion of AMP to adenosine. Kinetic constants for each reaction were estimated by fitting simulated reaction curves to observed time courses. Apparent Km values estimated in this way agreed well with those reported for initial velocity measurements (ATPase = 300 microM; ADPase = 240 microM; and 5'-nucleotidase = 26 microM). The ratio of maximum velocities was ATPase:ADPase:AMPase = 6:1.5:1, with absolute values varying among cell batches. The data could only be fitted if the model incorporated inhibition of 5'-nucleotidase by ATP or ADP, and satisfactory fitting was achieved with a Ki value for ADP of 5 microM. These kinetic properties maximize the time separation of the intermediate pools. In vivo, at sites of platelet degranulation, they would create a time gap proportional to the size of the initial release between release of ADP (a proaggregatory milieu) and the appearance of adenosine (an anti-aggregatory milieu).