Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Synthesis and biological activity of linear and cyclic enkephalins modified at the Gly3-Phe4 amide bond

As part of our continuing effort to define structure-activity relationships for enkephalin and design enzymatically resistant analogs, we report the synthesis and biological activities of linear and cyclic enkephalin analogs modified at the Gly3-Phe4 amide bond. The partial retro-inverso enkephalin analog Tyr-D-Ala-gGly-(R,S)-mPhe-Leu-NH2 and its cyclic counterpart, Tyr-cyclo[D-A2 bu-gGly-(R,S)-mPhe-Leu-], were synthesized as diastereomeric mixtures using solution methodology. The racemic benzylmalonate allowed the linear analog to be synthesized by fragment coupling at the reversed bond. Cyclization of the second analog was carried out at high concentration, eliminating formation of polymer by the use of an insoluble base. All gem-diaminoalkyl residues were prepared by conversion of peptidyl amides with benzene iodonium bis(trifluoroacetate). Diastereomers of both compounds were separable by reverse phase HPLC but those of the linear compound racemized rapidly under conditions of testing and were therefore tested together. All analogs tested had activities ranging from 6 to 14% of the activity of Leu enkephalin, indicating that the Gly3-Phe4 amide bond is important, though not crucial, for receptor binding.     

Monoclonal antibodies as probes of the alpha-bungarotoxin and cholinergic binding regions of the acetylcholine receptor

We have probed the acetylcholine receptor (AcChR) molecule with six anti-AcChR monoclonal antibodies (mAbs) whose binding to the AcChR is inhibited or blocked by alpha-bungarotoxin (alpha BgTx). mAbs bound with a maximum stoichiometry of either one mAb (387D, 247G) or two mAbs (383C, 572C, 370C, 249E) per AcChR monomer, and the extent to which they inhibited alpha BgTx binding directly correlated with their stoichiometry of binding. The effect of mAbs on the alpha BgTx and cholinergic ligand binding properties of the AcChR molecule defined three major categories of mAbs: those that block alpha BgTx and carbamylcholine (agonist) binding, but do not block d-tubocurarine (antagonist) binding (383C, 572C, 370C and 249E); mAb 387D, which blocks agonist binding and partially blocks alpha BgTx and d-tubocurarine binding; and mAb 247G, which does not affect agonist binding, blocks at most 50% of the alpha BgTx binding sites, and decreases the affinity of the high affinity component of d-tubocurarine binding (Mihovilovic, M., and Richman, D. P. (1984) J. Biol. Chem. 259, 15051-15059). Except for mAb 247G, these mAbs strongly competed with each other for binding to the AcChR. In contrast, mAb 247G blocks about 50% of the binding of all the other mAbs. The results demonstrate the ability of mAbs to stabilize different conformational states of the AcChR and to probe cholinergic epitopes of functional importance. They also indicate the nonequivalence of the two alpha-toxin binding regions of the AcChR molecule and suggest that it is possible to identify epitopes within the alpha BgTx binding region that when bound produce differential effects on the binding of the agonist (carbamylcholine) and the antagonist (d-tubocurarine).     

Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis

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The murine Kupffer cell. I. Characterization of the cell serving accessory function in antigen-specific T cell proliferation.

Murine Kupffer cells, the tissue macrophages of the liver, were isolated by collagenase digestion, differential sedimentation over Metrizamide, and glass adherence. The resultant cell population was more than 86% phagocytic, and 95% of cells stained positively for alpha-naphthyl butyrate esterase activity. The cells also had cell surface receptors for complement (C) and the Fc portion of IgG. In addition, a large proportion of Kupffer cells was shown to bear Ia antigens: about half of the cells bore I-A subregion-encoded antigens and about half bore I-BJE or I-EC subregion-encoded antigens. Kupffer cell populations were capable of reconstituting antigen-stimulated proliferative responses of antigen-primed, macrophage-depleted, lymph node T cells. The ability to reconstitute proliferation was enriched in the adherent population and was resistant to radiation and treatment with an anti-Thy antiserum and C. We conclude that isolated murine Kupffer cells bear the Ia phenotype of accessory cells that function in antigen presentation and that Kupffer cells can participate in the induction of antigen-specific immune responses. These data suggest that Kupffer cells may play a role in modulating responses to enterically derived antigens.     

Associations between virologic and immunologic dynamics in blood and in the male genital tract

To determine the influence of asymptomatic genital viral infections on the cellular components of semen and blood, we evaluated the associations between the numbers and activation statuses of CD4⁺ and CD8⁺ T lymphocytes in both compartments and the seminal levels of cytomegalovirus (CMV), herpes simplex virus (HSV), and human immunodeficiency virus 1 (HIV). Paired blood and semen samples were collected from 36 HIV-infected antiretroviral-naïve individuals and from 40 HIV-uninfected participants. We performed multiparameter flow cytometry analysis (CD45, CD45RA, CD3, CD4, CD8, and CD38) of seminal and blood cellular components and measured HIV RNA and CMV and HSV DNA levels in seminal and blood plasma by real-time PCR. Compared to HIV-uninfected participants, in the seminal compartment HIV-infected participants had higher levels of CMV (P < 0.05), higher numbers of total CD3⁺ (P < 0.01) and CD8⁺ subset (P < 0.01) T lymphocytes, and higher CD4⁺ and CD8⁺ T lymphocyte activation (RA-CD38⁺) (P < 0.01). Seminal CMV levels positively correlated with absolute numbers of CD4⁺ and CD8⁺ T cells in semen (P < 0.05) and with the activation status of CD4⁺ T cells in semen and in blood (P < 0.01). HIV levels in semen (P < 0.05) and blood (P < 0.01) were positively associated with T-cell activation in blood. Activation of CD8⁺ T cells in blood remained an independent predictor of HIV levels in semen in multivariate analysis. The virologic milieu in the male genital tract strongly influences the recruitment and activation of immune cells in semen and may also modulate T-cell immune activation in blood. These factors likely influence replication dynamics, sexual transmission risk, and disease outcomes for all three viruses.     

Use of Neurospora spore killer strains to obtain centromere linkage data without dissecting asci

Use of a centromere-linked Spore killer gene Sk reduces manyfold the labor involved in obtaining tetrad data that would otherwise require ordered dissection of intact linear eight-spored asci. Heterozygous crosses are made for Spore killer (SkK X SkS) and for markers to be tested. In such crosses only SkK ascospores survive. The four viable (SkK) and four aborted (SkS) ascospores of each ascus are ejected from the perithecium as a physically disordered group. The four surviving SkK ascospores of individual asci are germinated and scored. SkK segregates from SkS at the first meiotic division. If both marker alleles are represented in the surviving products, they must therefore have segregated from one another at the second division. Four-spore (Fsp) genes have been used to eliminate one postmeiotic nuclear division, so that only two ascospores per ascus need to be scored. The Spore killer method has been useful for mapping closely linked genes in centromere regions, for identifying genes that are far out on chromosome arms, for obtaining information on meiotic crossing-over, and for comparing linkages in different species.     

An immunofluorescence study of chondrogenesis in murine mandibular ectomesenchyme

The temporal and spatial distribution of type I collagen, type II collagen, cartilage-specific proteoglycan (CSPG) and fibronectin in mouse mandible is described. CD-1 mouse embryos of 12-, 15-, and 18-day gestation were used, and matrix molecules were localized using indirect immunofluorescence. On day 12, accumulation of type II collagen, CSPG, and fibronectin within regions of condensed mesenchyme was noted. On day 15, intense staining for type II collagen and CSPG occurred. Fibronectin was less brilliant with its greatest concentration near the perichondrium. On day 18, the cartilage matrix was undergoing osseous replacement concurrent with loss of type II collagen and CSPG. Type I collagen was seen in the perichondrium, membranous bone and sub-basement membrane region in specimens of all ages. Synthesis and expression of extracellular matrix molecules reflect patterns of differentiation in mandibular mesenchyme.