A comparison of the behavior of cholesterol and selected derivatives in mixed sterol-phospholipid Langmuir monolayers: a fluorescence microscopy study

Eukaryotic cells require sterols to achieve normal structure and function of their plasma membranes, and deviations from normal sterol composition can perturb these features and compromise cellular and organism viability. The Smith-Lemli-Opitz syndrome (SLOS) is a hereditary metabolic disease involving cholesterol (CHOL) deficiency and abnormal accumulation of the CHOL precursor, 7-dehydrocholesterol (7DHC). In this study, the interactions of CHOL and the related sterols desmosterol (DES) and 7DHC with l-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers were compared. Pressure-area isotherms and fluorescence microscopy were used to study DPPC monolayers containing 0, 10, 20, or 30 mol% sterol. Similar behavior was noted for CHOL- and DES-containing DPPC monolayers with both techniques. However, while 7DHC gave isotherms similar to those obtained with the other sterols, microscopy indicated limited domain formation with DPPC, indicating that 7DHC packs somewhat differently in DPPC membranes compared to CHOL and DES. These results are discussed in relation to SLOS pathobiology.     

Expression profiling of murine double-negative regulatory T cells suggest mechanisms for prolonged cardiac allograft survival

Recent studies have demonstrated that both mouse and human alpha beta TCR(+)CD3(+)NK1.1(-)CD4(-)CD8- double-negative regulatory T (DN Treg) cells can suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells. To identify molecules involved in DN Treg cell function, we generated a panel of murine DN Treg clones, which specifically kill activated syngeneic CD8+ T cells. Through serial cultivation of DN Treg clones, mutant clones arose that lost regulatory capacity in vitro and in vivo. Although all allogeneic cardiac grafts in animals preinfused with tolerant CD4/CD8 negative 12 DN Treg clones survived over 100 days, allograft survival is unchanged following infusion of mutant clones (19.5 +/- 11.1 days) compared with untreated controls (22.8 +/- 10.5 days; p < 0.001). Global gene expression differences between functional DN Treg cells and nonfunctional mutants were compared. We found 1099 differentially expressed genes (q < 0.025%), suggesting increased cell proliferation and survival, immune regulation, and chemotaxis, together with decreased expression of genes for Ag presentation, apoptosis, and protein phosphatases involved in signal transduction. Expression of 33 overexpressed and 24 underexpressed genes were confirmed using quantitative real-time PCR. Protein expression of several genes, including Fc epsilon RI gamma subunit and CXCR5, which are >50-fold higher, was also confirmed using FACS. These findings shed light on the mechanisms by which DN Treg cells down-regulate immune responses and prolong cardiac allograft survival.     

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2-MSH3 or MSH2-MSH6) or crossing over (MSH4-MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4-MSH5. The second complex involves MLH3 together with MSH2-MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2-/- males, but not females, providing an explanation for the sexual dimorphism seen in Pms2-/- mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2-MSH3 and is decreased in Msh2-/- and Msh3-/- mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.     

Mutation of the priA gene of Neisseria gonorrhoeae affects DNA transformation and DNA repair

In Escherichia coli, PriA is central to the restart of chromosomal replication when replication fork progression is disrupted and is also involved in homologous recombination and DNA repair. To investigate the role of PriA in recombination and repair in Neisseria gonorrhoeae, we identified, cloned, and insertionally inactivated the gonococcal priA homologue. The priA mutant showed a growth deficiency and decreased DNA repair capability and was completely for deficient in DNA transformation compared to the isogenic parental strain. The priA mutant was also more sensitive to the oxidative damaging agents H2O2 and cumene hydroperoxide compared to the parental strain. These phenotypes were complemented by supplying a functional copy of priA elsewhere in the chromosome. The N. gonorrhoeae priA mutant showed no alteration in the frequency of pilin antigenic variation. We conclude that PriA participates in DNA repair and DNA transformation processes but not in pilin antigenic variation.     

Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer

Protein labeling with green fluorescent protein derivatives has become an invaluable tool in cell biology. Protein quantification, however, is difficult when cells express constructs with overlapping fluorescent emissions. Under these conditions, signal separation using emission filters is inherently inefficient. Spectral imaging solves this problem by recording emission spectra directly. Unfortunately, linear unmixing, the algorithm used for quantifying individual fluorophores from emission spectra, fails when resonance energy transfer (RET) is present. We therefore sought to develop an unmixing algorithm that incorporates RET. An equation for spectral emission incorporating RET was derived and an assay based on this formalism, spectral RET (sRET), was developed. Standards with defined RET efficiencies and with known Cerulean/Venus ratios were constructed and used to test sRET. We demonstrate that sRET analysis is a comprehensive, photon-efficient method for imaging RET efficiencies and accurately determines donor and acceptor concentrations in living cells.     

The low-density lipoprotein receptor-related protein-1 associates transiently with lipid rafts

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional receptor that undergoes constitutive endocytosis and recycling. To identify LRP-1 in lipid rafts, we biotin-labeled cells using a membrane-impermeable reagent and prepared Triton X-100 fractions. Raft-associated proteins were identified in streptavidin affinity-precipitates of the Triton X-100-insoluble fraction. PDGF beta-receptor was identified exclusively in lipid rafts, whereas transferrin receptor was excluded. LRP-1 distributed partially into rafts in murine embryonic fibroblasts (MEFs) and HT 1080 cells, but not in smooth muscle cells and CHO cells. LRP-1 partitioning into rafts was not altered by ligands, including alpha2-macroglobulin, platelet-derived growth factor-BB, and receptor-associated protein (RAP). To examine LRP-1 trafficking between membrane microdomains, we developed a novel method based on biotinylation and detergent fractionation. Association of LRP-1 with rafts was transient; by 15 min, nearly all of the LRP-1 that was initially raft-associated exited this compartment. LRP-1 in the Triton X-100-soluble fraction, which excludes lipid rafts, demonstrated complex kinetics, with phases reflecting import from rafts, endocytosis, and recycling. Potassium depletion blocked LRP-1 endocytosis but did not inhibit trafficking of LRP-1 from rafts into detergent-soluble microdomains. Our data support a model in which LRP-1 transiently associates with rafts but does not form a stable pool. Fluid movement of LRP-1 between microdomains may facilitate its function in promoting the endocytosis of other plasma membrane proteins, such as the urokinase receptor, which localizes in lipid rafts.     

Synthesis and characterization of mPEG-PLA prodrug micelles

Polymeric prodrugs of mPEG-PLA-haloperidol (methoxypoly(ethylene glycol)-b-poly(lactic acid)) can self-assemble into nanoscale micelle-like structures in aqueous solutions. mPEG-PLA-haloperidol was prepared and characterized using 1H and 13C NMR. The conjugation efficiency was found to be 64.8 +/- 21%. Micelles that form spontaneously upon solubilization of the mPEG-PLA and the polymeric prodrugs in water were characterized using a variety of techniques. The mPEG-PLA and prodrug micelles were found to have diameters of 28.73 +/- 1.45 and 49.67 +/- 4.29 nm, respectively, using dynamic light scattering (DLS). The micelle size and polydispersity were also evaluated with cryogenic transmission electron microscopy (cryo-TEM) and were consistent with the DLS results. Cryo-TEM and proton NMR confirmed that the micelles were spherical in shape. DLS was also used to determine the aggregation numbers of the micelles. The aggregation numbers ranged from 351 to 603. The change in aggregation number was dependent on the total drug incorporation into the micelle core. Critical micelle concentrations were determined for the various micelle/drug formulations and found to range from 3 to 14 microg/mL. Finally, drug was incorporated into the micelle core using the conjugate, free drug with a saturated aqueous phase during production, or a combination of both techniques. Drug incorporation could be increased from 3% to 20% (w/w) using the different formulations.     

Recognition of fresh human tumor by human peripheral blood lymphocytes transduced with a bicistronic retroviral vector encoding a murine anti-p53 TCR

The p53 protein is markedly up-regulated in a high proportion of human malignancies. Using an HLA-A2 transgenic mouse model, it was possible to isolate high-avidity murine CTLs that recognize class I-restricted human p53 epitopes. We isolated the alpha- and beta-chain of a TCR from a highly avid murine CTL clone that recognized the human p53(264-272) epitope. These genes were cloned into a retroviral vector that mediated high efficiency gene transfer into primary human lymphocytes. Efficiencies of >90% for gene transfer into lymphocytes were obtained without selection for transduced cells. The p53 TCR-transduced lymphocytes were able to specifically recognize with high-avidity, peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with either wild-type or mutant p53 protein. p53 TCR-transduced cells demonstrated recognition and killing of a broad spectrum of human tumor cell lines as well as recognition of fresh human tumor cells. Interestingly, both CD8+ and CD4+ subsets were capable of recognizing and killing target cells, stressing the potential application of such a CD8-independent TCR molecule that can mediate both helper and cytotoxic responses. These results suggest that lymphocytes genetically engineered to express anti-p53 TCR may be of value for the adoptive immunotherapy of patients with a variety of common malignancies.     

THE ANATOMY OF SECRETION IN THE FOLLICULAR CELLS OF THE THYROID GLAND : II. The Effect of Acute Thyrotrophic Hormone Stimulation on the Secretory Apparatus

This paper reports a study by phase contrast and electron microscopy of changes observed in the thyroid gland of the rat at 1, 2, 12, and 24 hours following an injection of thyrotrophic hormone. Examination by phase contrast microscopy reveals that follicular cells contain numerous colloid droplets 1 and 2 hours after injection. By 12 and 24 hours, the colloid droplets are no longer present, and individual follicles appear to be subdividing into smaller units. The droplets are assumed to contain newly synthesized colloid, and their development was studied by electron microscopy. During the period of active secretion, increase in number of the Golgi vesicles leads to enlargement of this organelle. At its periphery small colloid droplets appear to form from large Golgi vesicles. As they form, their content becomes more adielectronic, and fine dense particles less than 75 A in diameter appear in their matrix. Small- and medium-sized droplets lying in the apical region of the cell contain numerous dense particles scattered in their moderately adielectronic content. Large, mature droplets in the same region have a relatively dielectronic content resembling follicular colloid and no longer contain dense particles. The follicular cells appear to utilize apical pseudopodia to release the content of mature droplets into the follicular lumen. Other droplet-like inclusions occur in follicular cells, but they do not seem to be directly concerned with secretion.