Topographic changes of membrane receptors for wheat germ agglutinin during the cell cycle and their relation to cytolysis

Experiments with synchronous cultures of murine mastocytoma cells indicated that cells incubated with wheat germ agglutinin (WGA) responded with cap formation and cytolysis during the mitotic phase only, but not during the G1, S and G2 phases of the cell cycle. The telophase of mitosis was found to be most sensitive to the cytolytic effect of WGA. Addition of the lectin to cells enhanced movement of the lectin-binding sites to the cleavage furrow during the telophase, as demonstrable by the concentration of fluorescent lectin and microvilli in this region. This phenomenon was followed by osmotic lysis of the cells which could be prevented by addition to the medium of dextran of high molecular weight.     

Cation binding to membranes: Competition between mono-, di- and trivalent cations

The binding of mono-, di- and trivalent cations to negatively charged surfaces is studied within the framework of a modified Gouy-Chapman equation. For any given combination of ions of the above valences, the existence and uniqueness of the solution for the surface potential is shown. The treatment provides the surface potential and charge density. For a system containing only monovalent and divalent ions, analytical solutions are given. When trivalent ions are also present, a procedure based on numerical integration is described. The distance dependence of the electrostatic potential for planar surfaces is given. The calculations provide the amount of cations tightly bound and the amount trapped in the double layer region. The competition between cations for binding to surfaces is elucidated.     

Specificity of the cholesterol side-chain cleavage enzyme system of adrenal cortex

Incubation of lanosta-8, 24-dien-3β-o1-1,2- ³H and lanost-8-en-3β-o1-1, 2-³H with an adrenocortical bovine mitochondrial acetone-dried preparation did not yield any significant ( < 0.01%) 3β-hydroxy-4, 4, 14-trimethyl-5α-pregn-8-en-20-one. Under the same conditions cholesterol-1,2-³H yielded 8.3% pregnenolone. Incubation of (20S?) — 17α, 20-dihydroxycholesterol-7-³ H yielded 0.6 to 1.6% (20SS?, 22R?) — 17α, 20, 22-trihydroxycholesterol, 1.0 to 3.2% of 17α-hydroxypregnenolone, but no significant ( < 0.02%) (20S, 22S)-17α, 20, 22-trihydroxycholesterol. In another experiment incubation of cholesterol-1, 2-³H yielded 5% pregnenolone, 0.5% 17α-hydroxypregnenolone, 0.2% (20R?,22R?)-20, 22-dihydroxy-cholesterol, but no significant ( < 0.01%) 17α-hydroxy-cholesterol, (20S?) -17α, 20-dihydroxycholesterol or (20S?, 22R?)-17α, 20, 22-trihydroxycholesterol.     

Distribution of cholesterol between the outer and inner halves of the lipid bilayer of mycoplasma cell membranes

Rapid kinetic studies of filipin binding to intact cells and isolated membranes were performed with a stopped-flow apparatus to determine the distribution of cholesterol in the outer and inner surfaces of mycoplasma membranes. The initial rates of association of filipin with cholesterol in Mycoplasma gallisepticum and Mycoplasma capricolum intact cells were slower than those obtained with isolated membrane preparations. Ratios of the second-order rate constants for filipin binding to cells relative to membranes indicate that cholesterol is distributed symmetrically in membranes of M. gallisepticum cells whereas in M. capricolum ～66% of the free cholesterol is localized in the outer half of the lipid bilayer.     

Noncollagenous bone proteins in experimental rickets in the rat

Rats were raised in the absence of vitamin D in utero and throughout post-fetal life and neither 1,25-dihydroxyvitamin D3 nor related metabolites were detected in serums. No changes were observed in the relative amount of extractable noncollagenous bone proteins (NCP) in rachitic compared to vitamin-D-repleted animals. As expected, the relative levels of the mineral-bound, serum-derived albumin and 2-HS glycoprotein were unaffected in bones of rachitic animals. Interestingly, the vitamin D deficiency also did not have dramatic effects on several bone cell-derived noncollagenous proteins including: bone proteoglycans I & 11, bone sialoprotein li osteonectin, and osteocalcin. In contrast to the proteoglycans, the bone sialoprotein II and osteonectin were found in the nonmineral compartment of the rachitic animals, presumably bound to the wide osteoid seam.     

Malic enzyme 2 may underlie susceptibility to adolescent-onset idiopathic generalized epilepsy

Idiopathic generalized epilepsy (IGE) is a class of genetically determined, phenotypically related epilepsy syndromes. Linkage analysis identified a chromosome 18 locus predisposing to a number of adolescent-onset IGEs. We report a single-nucleotide polymorphism (SNP) association analysis of the region around the marker locus with the high LOD score. This analysis, which used both case-control and family-based association methods, yielded strong evidence that malic enzyme 2 (ME2) is the gene predisposing to IGE. We also observed association among subgroups of IGE syndromes. An ME2-centered nine-SNP haplotype, when present homozygously, increases the risk for IGE (odds ratio 6.1; 95% confidence interval 2.9-12.7) compared with any other genotype. Both the linkage analysis and the association analysis support recessive inheritance for the locus, which is compatible with the fact that ME2 is an enzyme. ME2 is a genome-coded mitochondrial enzyme that converts malate to pyruvate and is involved in neuronal synthesis of the neurotransmitter gamma-aminobutyric acid (GABA). The results suggest that GABA synthesis disruption predisposes to common IGE and that clinical seizures are triggered when mutations at other genes, or perhaps other insults, are present.     

Cutting edge: Membrane nanotubes connect immune cells

We present evidence that nanotubular highways, or membrane nanotubes, facilitate a novel mechanism for intercellular communication in the immune system. Nanotubes were seen to connect multiple cells together and were readily formed between a variety of cell types, including human peripheral blood NK cells, macrophages, and EBV-transformed B cells. Nanotubes could be created upon disassembly of the immunological synapse, as cells move apart. Thus, nanotubular networks could be assembled from transient immunological synapses. Nanotubes were seen to contain GFP-tagged cell surface class I MHC protein expressed in one of the connected cells. Moreover, GPI-conjugated to GFP originating from one cell was transferred onto the surface of another at the connection with a nanotube. Thus, nanotubes can traffic cell surface proteins between immune cells over many tens of microns. Determining whether there are physiological functions for nanotubes is an intriguing new goal for cellular immunology.     

Translocation of histone proteins across lipid bilayers and Mycoplasma membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).