Cross-tissue immune cell analysis reveals tissue-specific features in humans.

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. We surveyed the immune compartment of 16 tissues from 12 adult donors by single-cell RNA sequencing and VDJ sequencing generating a dataset of ~360,000 cells. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of finely phenotyped immune cell types, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. Our multitissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis, and antigen receptor sequencing.

Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia.

The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR-HMP (cadherin-catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (alpha-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans.

Surface enhanced Raman scattering of bacteria using capped and uncapped silver nanoparticles.

Surface enhanced Raman scattering (SERS) spectra of bacteria were obtained using citrate (capped) and borohydride (uncapped) generated silver nanoparticles (Ag NPs).The observed differences in SERS spectra are attributed to the manner in which these Ag NPs interact with bacteria. Capped Ag NPs are able to partition through the surface polysaccharides of the bacterial cell to bind to the inner and outer cell membranes, as well as the periplasmic space between them. The resultant spectra show contributions due to the components of the cell envelope and cellular secretions. Uncapped Ag NPs are unable to partition through the polysaccharide outer structures of the cells. Spectral features observed for these uncapped Ag NPs are secretions primarily due to the metabolites of purine degradation.

Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology.

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosylphosphorylcholine (C(6)-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca(2+) ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-beta-cyclodextrin, which is indicative of a loss of tight SM-cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C(6)- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.

Epoxides of carcinogenic polycyclic hydrocarbons are frameshift mutagens.

K-region epoxides of the carcinogens benz[a] anthracene, dibenz[a,h]anthracene, and 7-methylbenz[a] anthracene are mutagenic in strains of Salmonella typhimurium designed to detect frameshift mutagens. Parent hydrocarbons, K-region diols and phenols and some other epoxides are inactive as mutagens in these tests. Polycyclic hydrocarbon epoxides, and other presumed proximal carcinogens, are discussed as examples of intercalating agents with reactive side chains. It has been shown previously that intercalating agents with reactive side chains are potent frameshift mutagens.

Overlapping but nonidentical binding sites on CD2 for CD58 and a second ligand CD59.

The interaction of the T cell glycoprotein CD2 with one ligand, CD58, contributes to T cell function. We have identified CD59, a glycoprotein with complement-inhibitory function, as a second physiological ligand for CD2. Antibodies to CD59 inhibit CD2-dependent T cell activation in murine T cell hybridomas expressing human CD2. In an in vitro binding assay with purified CD58 and CD59, CD2+ cells bind not only immobilized CD58 but also CD59. With two complementary approaches, it was demonstrated that the binding sites on CD2 for CD58 and CD59 are overlapping but nonidentical. These observations suggest that direct interactions between CD2 and both CD58 and CD59 contribute to T cell activation and adhesion.

Inactivation of a diol epoxide by dihydrodiol dehydrogenase but not by two epoxide hydrolases.

The mutagenicity of r-8,t-9-dihydroxy-t-10, 11-oxy-8,9,10,11-tetrahydrobenz[a]anthracene (BA-8,9-diol 10, 11-oxide) toward Salmonella typhimurium TA 100 is not decreased by the presence of large amounts of highly purified microsomal or cytosolic epoxide hydrolase. However, highly purified dihydrodiol dehydrogenase inactivates this diol epoxide, which is a major DNA-binding metabolite of benz[a]anthracene. The K-region epoxide, benz[a]anthracene 5,6-oxide (BA 5,6-oxide) is efficiently inactivated by microsomal epoxide hydrolase, is much less readily inactivated by cytosolic epoxide hydrolase, and is not inactivated by dihydrodiol dehydrogenase. This inactivation of a diol epoxide by dihydrodiol dehydrogenase points to a new significance of this enzyme and a new level of control for diol epoxides.

Antileukemic roles of human phospholipid scramblase 1 gene, evidence from inducible PLSCR1-expressing leukemic cells.

Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated protein which is localized in either the cell membrane or nucleus depending on its palmitoylated state. The increasing evidence showed the biological roles of PLSCR1 in cell signaling, maturation and apoptosis. To investigate the functions of PLSCR1 in leukemic cells, we generated an inducible PLSCR1-expressing cell line using myeloid leukemic U937 cells. In this cell line, PLSCR1 was tightly regulated and induced upon tetracycline withdrawal. Our results showed that inducible PLSCR1 expression arrested the proliferation of U937 cells at G1 phase. Meanwhile, PLSCR1-overexpressing U937 cells also underwent granulocyte-like differentiation with increased sensitivity to etoposide-induced apoptosis. Furthermore, we also found that PLSCR1 induction increased cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1) proteins, together with downregulation of S phase kinase-associated protein 2 (SKP2), an F-box subunit of the ubiquitin-ligase complex that targets proteins for degradation. Additionally, PLSCR1 induction significantly decreased c-Myc protein and antiapoptotic Bcl-2 protein. Although the exact mechanism by which PLSCR1 regulates these cellular events and gene expression remains unresolved, our results suggest that PLSCR1 plays the antagonistic role regarding leukemia development. These data will shed new insights into understanding the biochemical and biological functions of PLSCR1 protein.

The terminal complement proteins C5b-9 augment binding of high density lipoprotein and its apolipoproteins A-I and A-II to human endothelial cells.

Terminal complement protein complexes C5b-9 have been found in human atherosclerotic lesions. Insertion of C5b-9 in the endothelial cell membrane alters permeability, induces membrane vesiculation, and activates secretion. We hypothesized that complement might also alter interactions of the endothelial surface with lipoproteins, particularly high density lipoprotein (HDL), which is reported to inhibit C5b-9-induced hemolysis. We now demonstrate that exposure to C5b-9 increases (by 2- to 50-fold) specific binding of HDL and its apolipoproteins (apo) A-I and A-II to endothelial cells. Binding to cells exposed to antibody, C5b67, and C5b-8 was virtually unchanged. Enhanced binding was also dependent on the number of C5b-9 complexes deposited on the cells. Other agonists that activate endothelial secretion did not augment binding. Calcium was required for full exposure of new binding sites by C5b-9. The C5b-9-induced increase in binding was independent of the increase observed after cholesterol loading. In addition, apo A-I and A-II appear to compete for the same binding sites on untreated and C5b-9-treated cells. In contrast to the data reported for red cells, we were unable to detect significant inhibition of C5b-9-mediated endothelial membrane permeabilization by HDL (up to 1 mg/ml) or by apo A-I (up to 100 micrograms/ml). These data demonstrate that the C5b-9 proteins enhance endothelial binding of HDL and its apoproteins, suggesting that intravascular complement activation may alter cholesterol homeostasis in the vessel wall.

Changes in cytosolic Ca2+ associated with von Willebrand factor release in human endothelial cells exposed to histamine. Study of microcarrier cell monolayers using the fluorescent probe indo-1.

A method for measuring fluorescence in anchored monolayers of human endothelial cells is described and used to demonstrate changes in the cytosolic free-calcium concentration ([Ca2+]c) in these cells exposed to histamine and thrombin; some endothelial responses to both agonists (e.g., mitogenesis) have been suggested to be Ca2+-mediated. Umbilical vein endothelial cells were cultured on microcarriers and loaded with the Ca2+ indicator, indo-1. Enzymatic cell detachment was avoided by monitoring the indo-1 fluorescence ratio (400/480 nm) of a stirred suspension of cell-covered microcarriers. Basal [Ca2+]c was estimated to be 70-80 nM. Thrombin induced a transient dose-dependent increase in [Ca2+]c, which was active-site dependent. Histamine stimulated a dose-dependent increase in [Ca2+]c, which was reversed by removal of histamine and inhibited competitively by the H1-receptor antagonist pyrilamine, but not by the H2-receptor antagonist cimetidine. Furthermore, histamine induced a dose-dependent secretion of von Willebrand factor, which paralleled the rise in [Ca2+]c and was similarly blocked by the H1-receptor antagonist, and which may contribute to platelet deposition at sites of inflammation.

Characterization of the complement sensitivity of paroxysmal nocturnal hemoglobinuria erythrocytes.

The affected erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH II and PNH III cells) are abnormally sensitive to complement-mediated lysis. Normal human erythrocytes chemically modified by treatment with 2-amino-ethylisothiouronium bromide (AET) have been used as models for PNH cells inasmuch as they also exhibit an enhanced susceptibility to complement. To investigate the bases for the greater sensitivity of these abnormal cells to complement-mediated lysis, we compared binding of C3 and constituents of the membrane attack complex to normal, PNH II, PNH III, and AET-treated cells after classical pathway activation by antibody and fluid-phase activation by cobra venom factor complexes. When whole serum complement was activated by antibody, there was increased binding of C3 and C9 to PNH II, PNH III, and AET-treated cells, although the binding of these complement components to PNH II and PNH III cells was considerably greater than their binding to the AET-treated cells. In addition, all of the abnormal cell types showed a greater degree of lysis per C9 bound than did the normal erythrocytes. PNH III and AET-treated cells were readily lysed by fluid-phase activation of complement, whereas normal and PNH II erythrocytes were not susceptible to bystander lysis. The greater hemolysis of PNH III and AET-treated cells in this reactive lysis system was due to a quantitative increase in binding of constituents of the membrane attack complex. This more efficient binding of the terminal components after fluid-phase activation of whole serum complement was not mediated by cell-bound C3 fragments. These investigations demonstrate that the molecular events that characterize the enhanced susceptibility of PNH II, PNH III, and AET-treated erythrocytes to complement-mediated lysis are heterogeneous.

Production and characterization of transformed B-lymphocytes expressing the membrane defect of Scott syndrome.

Scott syndrome is a bleeding disorder associated with an isolated defect in expression of membrane coagulant activity by stimulated platelets. This defect represents a decrease in platelet membrane binding sites for coagulation factors Va and VIIIa, reflecting diminished surface exposure of phosphatidylserine (PS). To gain insight into the cellular and genetic basis for this disorder, B-lymphocytes from a patient with Scott syndrome and from normal donors were immortalized by EBV-transformation, and tested for their capacity to expose plasma membrane PS in response to the Ca2+ ionophore, A23187. Upon incubation with A23187, EBV-lymphoblasts derived from normal donors consistently induced surface expression of PS in > 70% of all cells, as detected by membrane association of the PS-binding proteins, factor Va or annexin V. PS exposure in these cells was maximal after 5 min, and saturated at < 100 microM external free [Ca2+]. By contrast, < 30% of Scott syndrome lymphoblasts exposed PS, and saturation was not observed at > 1 mM external free [Ca2+]. Single-cell clones derived from the Scott lymphoblasts all exhibited a diminished response to A23187 comparable with that of the parental cells, suggesting that all lymphocytes from this patient share this membrane abnormality. Hybridomas prepared by fusion of Scott lymphoblasts with the myeloma cell line UC-LUC showed responses to Ca2+ ionophore comparable to those observed for normal lymphoblasts and for hybridomas prepared by fusion of normal lymphoblasts with UC-LUC. This correction of the Scott abnormality suggests possible complementation of an aberrant gene(s) responsible for this disorder.

Scott syndrome erythrocytes contain a membrane protein capable of mediating Ca2+-dependent transbilayer migration of membrane phospholipids.

Phospholipid (PL) scramblase is a plasma membrane protein that mediates accelerated transbilayer migration of PLs upon binding Ca2+, facilitating rapid mobilization of phosphatidylserine to the cell surface upon elevation of internal Ca2+. In patients with Scott syndrome, a congenital bleeding disorder related to defective expression of membrane coagulant activity, circulating blood cells show decreased cell surface exposure of phosphatidylserine at elevated cytosolic [Ca2+], implying an underlying defect or deficiency of PL scramblase. To gain insight into the molecular basis of this disorder, we compared PL scramblase in Scott erythrocyte membranes to those of normal controls. Whereas membranes of Scott cells were unresponsive to Ca2+-induced activation of PL scramblase at neutral pH, apparently normal PL scramblase activity was induced at pH < 6.0. After extraction with octylglucoside, a membrane protein was isolated from the Scott cells which exhibited normal PL scramblase activity when reconstituted in vesicles with exogenous PLs. Like PL scramblase from normal erythrocytes, PL scramblase from Scott erythrocytes was maximally activated either by addition of Ca2+ (at pH 7.4) or by acidification to pH < 6.0, and similar apparent affinities for Ca2+ and rates of transbilayer transfer of PLs were observed. This suggests that the defect in Scott syndrome is related to an altered interaction of Ca2+ with PL scramblase on the endofacial surface of the cell membrane, due either to an intrinsic constraint upon the protein preventing interaction with Ca2+ in situ, or due to an unidentified inhibitor or cofactor in the Scott cell that is dissociated by detergent.

Inhibition of the complement membrane attack complex by the galactose-specific adhesion of Entamoeba histolytica.

The human complement system is an important early host defense against infection. Entamoeba histolytica activates the complement system but is resistant to killing by complement C5b-9 complexes deposited on the membrane surface. Our aim was to identify components of the amebic plasma membrane that mediate resistance to human complement C5b-9 by screening for neutralizing monoclonal antibodies. A monoclonal antibody was identified that abrogated amebic resistance to C5b-9, and the mAb was shown to recognize the parasite's galactose-specific adhesin. The purified adhesin bound to C8 and C9 and conferred C5b-9 resistance to sensitive ameba upon reconstitution; these activities of the adhesin were inhibited by the antiadhesin mAb. The E. histolytica adhesin shared sequence similarities and antigenic cross-reactivity with CD59, a membrane inhibitor of C5b-9 in human blood cells, suggesting both molecular mimicry and shared complement-inhibitory functions.

SERS substrates fabricated using ceramic filters for the detection of bacteria: Eliminating the citrate interference.

It was found that spectra obtained for bacteria on SERS substrates fabricated by filtering citrate-generated Ag nanoparticles (NPs) onto rigid, ceramic filters exhibited peaks due to citrate as well as the bacteria. In many cases the citrate spectrum overwhelmed that of the bacteria. Given the simplicity of the method to prepare these substrates, means of eliminating this citrate interference were explored. It was found that allowing a mixture of bacteria suspension and citrate-generated Ag NPs to incubate prior to filtering onto the ceramic filter eliminated this interference.

In vitro malignant transformation of mouse fibroblasts by non-K-region dihydrodiols derived from 7-methylbenz(a)anthracene, 7,12-dimethylbenz(a)anthracene, and benzo(a)pyrene.

The 8,9-dihydrodiols of 7-methylbenz(a)anthracene and 7,12-dimethylbenz(a)anthracene and the 7,8-dihydrodiol of benzo(a)pyrene, which are non-K-region diols with adjacent olefinic double bonds that can be metabolized to diol-epoxides, were more active than the parent hydrocarbons in inducing malignant transformation of M2 mouse fibroblasts; a fourth non-K-region diol, the 9,10-dihydrodiol of benzo(a)pyrene was less active than benzo(a)pyrene. The related K-region dihydrodiols, which lack adjacent olefinic double bonds, and 6-hydroxybenzo(a)pyrene were inactive, 7,8-Dihydrobenzo(a)pyrene, a more potent carcinogen than the 9,10 isomer, induced malignant transformation, but the 9,10 isomer was inactive. Transformed cells with abnormal morphology yielded sarcomas on injection into isologous mice; treated but morphologically normal cells did not. These results support the role of diols and diol-epoxides in the metabolic activation of polycyclic hydrocarbons.

Comparison of the products of the reaction of 7-methylbenz(a)anthracene 5,6-oxide and RNA, with those formed in 7-methylbenz(a)anthracene-treated cells.

RNA was isolated by a phenol extraction method from mouse embryo cells treated in culture with either [G-3H]-7-methylbenz(a)anthracene or [G-3H]-7-methylbenz(a)anthracene 5,6-oxide (the K-region epoxide). The RNA was degraded to ribonucleosides, mixed with ultraviolet-absorbing quantities of the epoxide ribonucleoside products isolated from RNA that had reacted with 7-methylbenz(a)anthracene 5,6-oxide in aqueous ethanol solution, and chromatographed on a column of Sephadex LH-20 eluted with a methanol:water gradient. The 7-methyl-benz(a)anthracene 5,6-oxide ribonucleoside products formed in cells were identical to those formed in aqueous solution, although the relative amounts of the products varied. The majority of these epoxide-ribonucleoside products were not identical to the products formed in cells treated with the parent hydrocarbon. These results suggest that the major reactive form of 7-methylbenz(a)anthracene that binds to RNA in mouse embryo cells is not the K-region epoxide of this hydrocarbon.

Inactivation of a diol-epoxide and a K-region epoxide with high efficiency by glutathione transferase X.

Four glutathione transferases (EC 2.5.1.18), glutathione transferases A, B, and C and a hitherto unknown form, termed X, were purified to apparent homogeneity from rat liver cytosol. They were investigated for their abilities to inactivate two mutagenic epoxides derived from the polycyclic aromatic hydrocarbon benz(a)anthracene, the K-region epoxide benz(a)anthracene 5,6-oxide and the diol-epoxide r-8,t-9-dihydroxy-t-10,11-oxy-8,9,10, 11-tetrahydrobenz(a)anthracene. Mutagenic activity was determined using Salmonella typhimurium his- strain TA100. Glutathione alone had little if any influence on the mutagenicity of the diol-epoxide but significantly decreased the mutagenic effect of the K-region epoxide. This inactivation was enhanced by the addition of glutathione transferases. Both epoxides were inactivated by glutathione in the presence of each of the four enzymes, but with varying efficiencies. Inactivation of the K-region epoxide (in terms of its mutagenicity in the presence of glutathione) required extremely little enzyme, about 1000 times less than for the diol-epoxide. On a molar basis, glutathione transferase X (followed by C greater than A greater than or equal to B) was clearly the most efficient enzyme in inactivating both substrates and also more efficient than were three other purified enzymes (microsomal epoxide hydrolase, cytosolic epoxide hydrolase, and dihydrodiol dehydrogenase) previously investigated in this test system. Taking into account the amounts of enzyme present in rat liver, the glutathione transferases C and X were most effective in inactivating the epoxides examined. Thus, the newly discovered glutathione transferase X appears to be of substantial significance in the inactivation of two structural prototypes of epoxides derived from polycyclic aromatic hydrocarbons, a K-region epoxide and a non-bay-region vicinal diol-epoxide.

Phanerochaete chrysosporium and its natural substrate.

We seek to define more fully how Phanerochaete chrysosporium degrades its natural substrate, lignocellulose. This contribution concerns several relevant topics. Mineralisation of [14C]DHP, as a model for lignin degradation, showed that a set of genetically defined meiotically derived products of strain ME446 differed in their degradative ability and also that, under optimum conditions for mineralisation, extracellular lignin peroxidase activity was absent. Xylanolytic and xylosidase/beta(1-->3) glucanase activities are also described. The complexity of the CBHI gene family is described and differential splicing of a CBHI gene transcript is proposed. In contrast to the multiplicity of CHBI genes there is a single CBHII gene. PCR methods were developed to analyse differential gene expression on different substrates. We have also developed a transformation system involving a reporter construct for the analysis of CBHI promoter function.

Complement pores in erythrocyte membranes. Analysis of C8/C9 binding required for functional membrane damage.

The number of membrane-bound terminal complement proteins (C5b-9) required to generate a functional pore in the human erythrocyte membrane ghost has been determined. Resealed erythrocyte ghost membranes (ghosts) were treated with human complement proteins C5b6, C7, 131I-C8, and 125I-C9 under non-lytic conditions. Following C5b-9 assembly, sucrose-permeant ghosts were separated from C5b-9 ghosts that remained impermeant to sucrose by centrifugation over density barriers formed of 43% (w/v) sucrose. Analysis of 131I-C8 and 125I-C9 bound to sucrose-permeant and sucrose-impermeant subpopulations of C5b-9 ghosts revealed: 1. Sucrose-permeant C5b-9 ghosts show increased uptake of both 131I-C8 and 125I-C9 as compared to ghosts that remain impermeant to sucrose. Ghosts with less than 300 molecules 131I-C8 bound remain impermeant to sucrose, irrespective of the total C9 input, or, the multiplicity of C9 uptake by membrane C5b-8. 2. In the presence of excess 125I-C9, the ratio of 125I-C9/131I-C8 bound to membrane C5b67 is 3.2 +/- 0.8 (mean +/- 2 S.D.), suggesting an average stoichiometry of 3 C9 per C5b-8. Under these conditions, the ratio of 125I-C9/131I-C8 bound to sucrose-permeant ghosts (3.3 +/- 0.7) does not significantly differ from the ratio bound to sucrose-impermeant ghosts (2.9 +/- 0.6). 3. With limiting C9 input, the threshold of total C5b-8 uptake required for sucrose permeability increases significantly above 300 per cell when the ratio of bound 125I-C9/131I-C8 is decreased below unity. In the complete absence of C9, 11 700 C5b-8 complexes are bound to sucrose-permeant ghosts. It is concluded that more than 300 C5b-9 complexes must bind to the human erythrocyte to form a sucrose-permeant lesion. Although the binding of one C9 per C5b-8 is critical to the pore-forming activity of these proteins, the binding of additional molecules of C9 to each complex (C9/C8 greater than 1) does not significantly alter the threshold of total C5b-9 uptake required for lesion formation.