External Surface Of Plasma Membrane Research Articles

Development of a Ten-lncRNA Signature Prognostic Model for Breast Cancer Survival: A Study with the TCGA Database.

Long noncoding RNA (lncRNA) plays a critical role in the development of tumors. The aim of our study was construction of a lncRNA signature model to predict breast cancer (BRCA) patient survival. We downloaded RNA-seq data and relevant clinical information from the Cancer Genome Atlas (TCGA) database. Differentially expressed lncRNA were computed using the “edgeR” package and subjected to the univariate and multivariate Cox regression analysis. Corresponding protein-coding genes were used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis. Finally, 521 differentially expression lncRNA were obtained. We constructed a ten-lncRNA signature model (LINC01208, RP5-1011O1.3, LINC01234, LINC00989, RP11-696F12.1, RP11-909N17.2, CTC-297N7.9, CTA-384D8.34, CTC-276P9.4, and MAPT-IT1) to predict BRCA patient survival using the multivariate Cox proportional hazard regression model. The C-index was 0.712, and AUC scores of training, test, and entire sets were 0.746, 0.717, and 0.732, respectively. Univariate Cox regression analysis indicated that age, tumor status, N status, M status, and risk score were significantly related to overall survival in patients with BRCA. Further, the multivariate analysis showed that risk score and M status had outstanding independent prognostic values, both with p < 0.001. The Gene Ontology (GO) function and KEEG pathway analysis was primarily enriched in immune response, receptor binding, external surface of plasma membrane, signal transduction, cytokine-cytokine receptor interaction, and cell adhesion molecules (CAMs). Finally, we constructed a ten-lncRNA signature model that can serve as an independent prognostic model to predict BRCA patient survival.

Ultrastructural Assessment of 2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide activity on human breast adenocarcinoma cells

The aim of the present study was to investigate ultrastructural changes induced by (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (APHCA) treatment on human breast adenocarcinoma cancer cells MCF-7, besides the evaluation of phosphatidylserine externalization and DNA fragmentation in treated cells. Cell viability analysis demonstrated concentration and time-manner cytotoxicity. Treated MCF-7 cells did not expose phosphatidylserine residues to the external plasma membrane surface and DNA fragmentation was not visualized by electrophoresis. Light microscopy showed compromised cell density and presence of vacuolization after APHCA treatment with 60μM. Scanning and transmission electron microscopies revealed hallmarks of autophagy, namely the presence of membrane bebbling and autophagosomes, besides shrunken cells and cell debris in treated MCF-7 cells. However, more specific tests such as the quantification of mammalian autophagy proteins are necessary to determine the kind of death that is trigged by APHCA.

Neuronal Death Induced by Nanomolar Amyloid β Is Mediated by Primary Phagocytosis of Neurons by Microglia

Alzheimer disease is characterized by neuronal loss and brain plaques of extracellular amyloid β (Aβ), but the means by which Aβ may induce neuronal loss is not entirely clear. Although high concentrations of Aβ (μM) can induce direct toxicity to neurons, we find that low concentration (nM) induce neuronal loss through a microglia-mediated mechanism. In mixed neuronal-glial cultures from rat cerebellum, 250 nM Aβ1-42 (added as monomers, oligomers or fibers) induced about 30% loss of neurons between 2 and 3 days. This neuronal loss occurred without any increase in neuronal apoptosis or necrosis, and no neuronal loss occurred with Aβ42-1. Aβ greatly increased the phagocytic capacity of microglia and induced phosphatidylserine exposure (an "eat-me" signal) on neuronal processes. Blocking exposed phosphatidylserine by adding annexin V or an antibody to phosphatidylserine or inhibiting microglial phagocytosis by adding either cytochalasin D (to block actin polymerization) or cyclo(RGDfV) (to block vitronectin receptors) significantly prevented neuronal loss. Loss of neuronal synapses occurred in parallel with loss of cell bodies and was also prevented by blocking phagocytosis. Inhibition of phagocytosis prevented neuronal loss with no increase in neuronal death, even after 7 days, suggesting that microglial phagocytosis was the primary cause of neuronal death induced by nanomolar Aβ.

Early developmental expression of a normally tumor-associated and drug-inhibited cell surface-located NADH oxidase (ENOX2) in non-cancer cells.

Full length mRNA to a drug-inhibited cell surface NADH oxidase, tNOX or ENOX2, is present in both non-cancer and cancer cells but is translated only in cancer cells as alternatively spliced variants. ENOX2 is a growth-related protein of the external plasma membrane surface that is shed into the circulation and is inhibited by a series of quinone site inhibitors with anticancer activity. To test the possibility that ENOX2 expression might be important to early stages of non-cancer cell development, the expression of the protein was monitored in chicken embryos during their development. Polyclonal antisera to a 34 kDa human serum form of ENOX2 cross-immunoreactive with the drug-responsive NADH oxidase of chicken hepatoma cells was used. The protein was identified based on drug-responsive enzymatic activities and analyses by western blots. The drug-responsive activity was associated with plasma membranes and sera of early chicken embryos and with chicken hepatoma plasma membranes but was absent from plasma membranes prepared from livers or from sera of normal adult chickens and from late embryo stages. The findings suggest that ENOX2 may fulfill some functions essential to the growth of early embryos which are lost in late embryo stages and absent from normal adult cells but which then reappear in cancer.

The plasma membrane-associated NADH oxidase (ECTO-NOX) of mouse skin responds to blue light

NADH oxidases of the external plasma membrane surface (ECTO-NOX proteins) are characterized by oscillations in activity with a regular period length of 24 min. Explants of mouse skin exhibit the oscillatory activity as estimated from the decrease in A 340 suggesting that individual ECTO-NOX molecules must somehow be induced to function synchronously. Transfer of explants of mouse skin from darkness to blue light (495 nm, 2 min, 50 μmol m −1 s −1) resulted in initiation of a new activity maximum (entrainment) with a midpoint 36 min after light exposure followed by maxima every 24 min thereafter. Addition of melatonin resulted in a new maximum 24 min after melatonin addition. The findings suggest that the ECTO-NOX proteins play a central role in the entrainment of the biological clock both by light and by melatonin.

Apoptosis and mitochondrial damage in INS-1 cells treated with alloxan.

To evaluate the participation of mitochondrial damage, oxygen radicals and cell death in diabetes mellitus, we designed a way to investigate INS-1 cells, rat pancreatic beta-cell line, to die by treatment with alloxan which generate reactive oxygen species (ROS). Incubation of INS-1 cells with alloxan for 24 h resulted in a decrease in viability of cells as well as inhibition of glucose-stimulated insulin release; this could be prevented by antioxidants, vitamin E and butylated hydroxyanisol (BHA). The formation of a DNA ladder and the distribution of phosphatidylserine at the external surface of plasma membrane were observed as indicators of apoptosis in the cells treated with alloxan at concentrations below 0.5 mM. The formation of DNA ladder was prevented by vitamin E, BHA and catalase, suggesting that the ROS is involved in the process of apoptosis in INS-1 cells treated with alloxan. Lower levels of intracellular ATP, collapse of mitochondrial membrane potential and release of cytochrome c from mitochondria were also observed in INS-1 cells treated with alloxan, suggesting that alloxan caused the damage of mitochondria in cells and was related to the process of apoptosis. In contrast, rat liver RLC-18 cells treated with alloxan were not observed in the decrease of viability. It follows from the present study that mitochondrial damages by ROS generated from alloxan is linked to apoptosis in INS-1 cells.

Occurrence of Amphoterin (HMG1) as an Endogenous Protein of Human Platelets that Is Exported to the Cell Surface upon Platelet Activation

Amphoterin (HMG1) is a 30-kD heparin-binding protein which is functionally associated with the outgrowth of cytoplasmic processes in developing neurones. Amphoterin has been shown to mediate adhesive and proteolytic interactions at the leading edge of motile cells. Recently it was shown that inhibition of amphoterin interactions with its cell surface receptor (RAGE) suppresses tumour growth and metastasis. In this work we have identified amphoterin polypeptide and its mRNA in human platelets. Amphoterin had a cytoplasmic localisation in resting platelets according to subcellular fractionation studies and immunogold electronmicroscopy. After platelet activation, part of amphoterin was associated with the external surface of plasma membrane. Externalisation of amphoterin during platelet activation was also detected in immunofluorescence studies. Amphoterin was detectable in human serum (0.2 ng/ml) but not in plasma. Resting platelets treated with PGI2 and forskolin bound to immobilised recombinant amphoterin independently of divalent cations. The binding induced a spicular morphology in platelets, and was effectively inhibited by heparin. Amphoterin-binding protein components on the platelet surface were not identified, but amphoterin bound to phosphatidylserine and sulfatide in lipid binding assays. Our results suggest that amphoterin is an endogenous protein in human platelets, which is exported to the cell surface during platelet activation. Interaction of amphoterin with the platelet surface may be mediated by sulfoglycolipids and phospholipids.

Use of dipyridyl-dithio substrates to measure directly the protein disulfide-thiol interchange activity of the auxin stimulated NADH: protein disulfide reductase (NADH oxidase) of soybean plasma membranes.

Dipyridyl-dithio substrates were cleaved by isolated vesicles of plasma membranes prepared from etiolated hypocotyls of soybean. The cleavage was stimulated by auxins at physiological concentrations. The substrates utilized were principally 2,2'-dithiodipyridine (DTP) and 6,6'-dithiodinicotinic acid (DTNA). The DTP generated 2 moles of 2-pyridinethione whereas the 6,6'-dithiodinicotinic acid generated 2 moles of 6-nicotinylthionine. Both products absorbed at 340 nm. The auxin herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) stimulated the activity approximately 2-fold to a maximum at about 10 microM. Concentrations of 2,4-D greater than 100 microM inhibited the activity. Indole-3-acetic acid stimulated the activity as well. The growth-inactive auxin, 2,3-dichlorophenoxyacetic acid (2,3-D), was without effect. DTNA cleavage correlated with oxidation of NADH and reduction of protein disulfide bonds reported earlier in terms of location at the external plasma membrane surface, absolute specific activity, pH dependence and auxin specificity. The dipyridyl-dithio substrates provide, for the first time, a direct measure of the disulfide-thiol interchange activity of the protein previously measured only indirectly as an auxin-dependent ability of isolated plasma membrane vesicles to restore activity to scrambled and inactive RNase.

A Single Amino Acid Difference within the Folate Transporter Encoded by the Murine RFC-1 Gene Selectively Alters its Interaction with Folate Analogues: IMPLICATIONS FOR INTRINSIC ANTIFOLATE RESISTANCE AND DIRECTIONAL ORIENTATION OF THE TRANSPORTER WITHIN THE PLASMA MEMBRANE OF TUMOR CELLS

The apparent Km, but not Vmax, for influx of methotrexate (MTX) mediated through the plasma membrane of S180 cells by the one-carbon, reduced folate transporter as well as the KD for binding to the transporter were 4-fold higher than in L1210 cells correlating with the greater intrinsic resistance of the former to this folate analogue. In contrast, no difference was observed between each cell type with regard to efflux of [3H]MTX mediated by this same transporter in ATP-depleted cells. The difference in influx Km in the case of this 10-methyl substituted N1O analogue of folic acid was not seen with more effective permeants, such as the unsubstituted N1O aminopterin or C1O analogues. Thus, values for influx Km for aminopterin, which were 1-1.2 microM in each cell type, increased as a result of substitution at N1O (MTX) 3-fold in L1210 cells but 12-fold in S180 cells. Nucleotide sequencing of reverse transcriptase-polymerase chain reaction-generated cDNA and of polymerase chain reaction-generated genomic DNA identified a single nucleotide difference between each cell type at +890 within exon 3 of the RFC-1 gene. This was in the form of a G (L1210 cells) to A (S180 cells) transition. Codon 297, the site of this transition, encodes either Ser or Asn in L1210 or S180 cells, respectively, which is located between the seventh and eight membrane-spanning helices. This amino acid difference had no effect on the electrophoretic mobility or amount of the transporter in each cell type that was shown by Western blotting with anti-RFC-1 peptide antibodies to migrate as 46 kDa in each case. Proof that this nucleotide difference alone accounted for the alteration in influx between each cell type was obtained by S180 RFC-1 cDNA versus L1210 RFC-1 cDNA transfection of an L1210 cell variant with undetectable MTX influx and RFC-1 gene expression. In this case, the higher Km for MTX influx associated with S180 cells was duplicated only in the S180 RFC-1 transfectants. These results appear to document the first example of a nucleotide alteration within the RFC-1 gene, which influences the interaction of MTX with the encoded plasma membrane transporter. An analysis of topology, in addition to other considerations, suggests that the site of the amino acid difference found in the transporter from L1210 and S180 cells occurs within or near the binding site on the external plasma membrane surface.

NADH oxidase activity present on both the external and internal surfaces of soybean plasma membranes

Soybean plasma membranes exhibit NADH oxidase activities accessible to NADH supplied to either the external or internal membrane surfaces. Activity at the external surface was demonstrated using plasma membranes of right side-out orientation prepared by aqueous two-phase partition and using intact soybean cells grown in culture. Activity at the internal membrane surface was demonstrated using vesicles of inside-out orientation obtained by preparative free-flow electrophoresis or by aqueous two-phase partition following freeze–thaw or Brij detergent treatment to invert some of the right side-out vesicles. The NADH oxidase activity of both membrane surfaces appeared to be stimulated by the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Following the freeze–thaw treatment utilized to evert the vesicles, however, the NADH oxidase activity of right side-out vesicles was greatly reduced. A 2,4-D-stimulated activity of these vesicles could be restored by treatment first with reduced glutathione and then with hydrogen peroxide or oxidized glutathione. Upon treatment with 0.1% Triton X-100, both right side-out and inside-out vesicles exhibited approximately the same specific activity and the same 2,4-D-stimulated component of activity equal to the sum of the activities of the right side-out and inside-out vesicles assayed separately. These findings suggest that a significant NADH oxidase activity of the soybean plasma membrane responsive to 2,4-D resides on the external plasma membrane surface. This latter activity is unlikely to function under physiological conditions in the direct oxidation of NADH and alternative functions, such as in protein disulfide–thiol interchange, would be more appropriate.

Impermeant antitumor sulfonylurea conjugates that inhibit plasma membrane NADH oxidase and growth of HeLa cells in culture. Identification of binding proteins from sera of cancer patients

The antitumor sulfonylurea LY237868 ( N-(4-aminophenyl-sulfonyl)- N′-(4-chlorophenyl)urea) was conjugated through the A ring to α-cyclodextrin or agarose bead material (Affigel 10) to prepare impermeant conjugates for activity measurements and affinity isolation of binding proteins from serum. When conjugated to α-cyclodextrin, the resulting LY237868 conjugate inhibited both NADH oxidase activity and growth of HeLa cells in culture. The conjugate was at least one order of magnitude more potent as an inhibitor than the parent compound. These findings confirm previous results that demonstrate an antitumor sulfonylurea-binding protein with NADH oxidase activity at the external plasma membrane surface of HeLa cells that is shed into culture media conditioned by growth of HeLa cells. A comparable activity, responsive to sulfonylurea, was present in sera of cancer patients. LY237868 conjugated to agarose beads as the affinity support bound a large number of serum proteins. However, compared to serum from normal patients, the affinity support bound two proteins of M r approx. 33.5 and 29.5 not found in sera of normal patients. The 33.5 kDa protein from human sera reacted with antisera to a 33.5 kDa protein from culture media conditioned by growth of HeLa cells that blocked and immunoprecipitated the sulfonylurea-responsive activity from HeLa cell plasma membranes. The results point to the 33.5 kDa protein from cancer patient sera that bound to the sulfonylurea affinity support as representing the circulating equivalent of the previously identified 34 kDa sulfonylurea-binding protein, with NADH oxidase activity at the external cell surface of cultured HeLa cells and a corresponding 33.5 kDa protein shed into culture media conditioned by growth of HeLa cells.

Histochemical methods for localizing and characterizing cell and tissue glycoconjugates

Glycoproteins, glycolipids and proteoglycans, collectively termed glycoconjugates, account for a large percentage of all tissue constituents. The carbohydrate moieties associated with these macromolecules have long been known or suspected to play specific biologic roles such as protection of the peptide backbone from recognition by antibodies and proteases, regulation of proper folding, assembly and exocytosis of secretory glycoproteins and lubrication of mucosal surfaces. Structural studies of complex carbohydrates have gained momentum over the past two decades with the realization that they participate in a much wider range of dynamic biological processes than was previously envisioned. Expressed predominantly at the external surface of plasma membranes and in the extracellular matrix glycoconjugates possessing unique saccharide sequences serve as recognition sites (ligands) for endogeneous or exogeneous carbohydrate binding proteins (lectins). Lectin-ligand interactions are involved in cell-cell and cell-substrate recognition events which occur for instance, during development and morphogenesis, wound healing and activation of immune responses.

Pharmacological study of tooth formation and mineralization using a tooth germ cultivation system

In this study, we introduce a cultivation method for mandibular first molars from mouse embryos. We also investigate the in vitro effects of several drugs on tooth formation. Histological observations demonstrated that the tooth germ dissected from 17-day-old embryonic mice and cultivated for 7 days under the conditions described here showed clear mineralization in the dentin and enamel. Alkaline phosphatase (ALP) activity and calcium content in the tooth germ increased developmentally during 7-day cultivation. The increase of calcium was preceded by that of ALP in the germ. The removal of ALP attached covalently to the external surface of plasma membranes by phosphatidylinositol specific phospholipase C disturbed in vitro mineralization in the tooth germ, suggesting that ALP functioning as an ectoenzyme is involved in the physiological process of tooth formation. To elucidate the effects of calcium regulating hormones and several drugs on tooth mineralization, ALP and calcium content were measured and also the tooth germ was observed histologically. The results obtained from the present study suggest that this in vitro system provides a useful tool for investigating both the direct action of drugs on tooth formation and the mechanisms of drug action.

Spontaneous interbilayer transfer of hexosyl ceramides between phospholipid bilayers

The kinetics of spontaneous transfer of various glucosyl- and galactosylceramides between 1-palmitoyl-2-oleoylphosphatidylcholine vesicles have been examined at 45 degrees C. Bovine brain galactosylceramides, kerasin and phrenosin, were found to transfer with biexponential kinetics. The kerasin fast pool is approximately 17% with a half-time of 29 h and the slow pool approximately 83% with a half-time of 2700 h. In contrast, semisynthetic N-palmitoylgalactosylceramide at the same temperature transfers with single-exponential kinetics with a half-time of 32 h. The half-time for N-lignoceroylgalactosylceramide under the same conditions proved to be greater than 3500 h. No concentration dependence for these half-times was found in the concentration range studied (0-10 mol%). Similar results were obtained for semisynthetic glucosylceramides. The biexponential kinetics observed for the two bovine brain ceramides, both of which are mixtures of short and long acyl chain molecules, are most probably a reflection of the strong dependence of transfer rate on acyl chain length. The very slow transfer rates of the long acyl chain hexosylceramides ensure that these molecules are lost very slowly, if at all, by spontaneous transfer from the external surface of plasma membranes; a result that is consistent with the putative biological role of glycosphingolipids.

The Distinctive Pattern of Proteoglycan and Glycosaminoglycan Free Chain Synthesis by Cultured Human Epidermal Keratinocytes

The in vitro synthesis of proteoglycans and glycosaminoglycan free chains was studied in human epidermal keratinocytes. Preconfluent and confluent cultures established on 3T3 feeders were steady state labeled with [35S]-sulfate and [3H]-glucosamine after removal of the 3T3 cells. Products in nonionic detergent extracts of keratinocytes and in the medium were analyzed in the presence of protease inhibitors. Glycosaminoglycans as proteoglycans and as free chains were defined by susceptibility or resistance, respectively, to alkaline borohydride reduction. Products associated with the cells were approximately 30% proteoglycans and approximately 70% glycosaminoglycan free chains, whereas in the medium virtually all was proteoglycan. The heparan and chondroitin sulfate proteoglycans were small compared to those of many other cell types. Their Kav on Sepharose CL-4B was 0.56 (estimated 50 kDa), whereas the free chain Kav was 0.74 (estimated 12 kDa). Relative amounts of the sulfated products varied with confluence and differentiation; heparan and chondroitin sulfates were equally represented within the free chains and proteoglycans of the cells in preconfluent, proliferating cultures, whereas in postconfluent, differentiated cultures the major labeling was in the heparan sulfate products, consistent with our prior reports (J Invest Dermatol 88:215-9, 1987 and 91:492-8, 1988). The cellular localization of the products was probed with glycosaminoglycan degrading enzymes added to isotopically prelabeled cultures. The proteoglycans appeared to be located on the external surface of plasma membranes, whereas the glycosaminoglycan free chains resisted digestion and are either intracellular or membrane associated, but otherwise inaccessible. These data establish the distinctive pattern of low Mr proteoglycans and abundant cell-associated glycosaminoglycan free chains synthesized by keratinocytes.

Detection of staphylococcal membrane receptors on virus-infected cells by direct adhesin overlay.

We partially characterized the interaction between 125I-labeled surface proteins of Staphylococcus aureus, obtained by thermal extraction, and purified plasma membranes from uninfected and influenza virus A/FM/1/47-infected Madin-Darby canine kidney cells. The radioactivity profile of surface-labeled proteins, derived from anion-exchange high-pressure liquid chromatography, showed a mixture of acidic polypeptides in a peak which contained approximately 5% of the total protein injected; adsorption with purified plasma membranes reduced radioactivity by 5 to 7% and altered elution profiles. Using a direct adhesin overlay procedure, we found that these surface-labeled proteins reacted with polypeptides located on the external surface of plasma membranes which were shared by both virus-infected and uninfected cells or were unique to virus-infected cells. Our data may help explain the enhanced adherence of S. aureus to influenza virus A-infected cells in vitro.

An ecto-ATPase of thyroidal cell membrane.

The isolated cells were obtained from hog thyroid glands treated with dispase. More than 95% of the cells obtained were intact and viable immediately after preparation, and the cell viability did not change during incubation in the experimental conditions. ATP added to the external medium of whole cell suspensions was hydrolyzed in the presence of various divalent cations, especially Mg, and the rate of hydrolysis of ATP was not significantly different between the Mg-ion system and the completed ion system (Mg+Na+K). When whole cell suspensions were disrupted with homogenizer, the hydrolysis of ATP was markedly increased by adding Na plus K. But there was no difference in the Mg-ion system between cell homogenates and whole cell suspensions. ADP, AMP and adenosine as reaction products were found in the reaction mixture which resulted from the hydrolysis of ATP by whole cell suspensions. Our data suggest that Mg-ATPase in the thyroidal isolated cells is an ectoenzyme whose active site(s) are exposed to the external surface of plasma membrane, and that ATP is finally hydrolyzed to adenosine via ADP and AMP by the enzyme(s).

Ultrastructural localization of alkaline phosphatase activity in sheep parathyroid gland

The histochemical localization of alkaline phosphatase in sheep parathyroid gland was investigated using light and electron microscopy. The reaction products of enzyme activity were observed by light microscopy in pericytes. By electron microscopy they were limited to the intercellular spaces between the gland cells, being exclusively confined to the external surface of plasma membrane.

Surface properties of plasma membranes following ionizing radiation exposure

Summary The physical properties of the external surface of plasma membranes of L5178Y mouse leukemic cells and cells from lines 26 and 37 derived from B16 mouse melanoma were studied in culture after exposure to 300 kVp X-rays. Measurements of cell surface charge: space relationships monitored by particle microelectrophoresis and partition in aqueous two-phase dextran-polyethylene glycol systems failed to disclose the massive decreases in surfaces charge described by other authors. Radio-induced cell synchronization, hydrogen peroxide formation effects, and sialic acid release from the cell surface have all proved negative in causing early irradiation-induced plasma membrane alterations. The relationship of electrophoretic mobility vs pH for irradiated cells suggests that the charge-determining moieties may not be as readily ionizable at low pH in the irradiated cell surfaces as in the control cell surfaces. The results are discussed in relation to cell membrane fluidity and programmed cell death.

Localization of tumour specific antigen on external surface of plasma membrane.

FOLLOWING the adventitious finding that lymphocytes from patients with malignant (but not benign) neoplasia are sensitized to encephalitogenic basic protein of myelin (EF)1 and to a protein similarly extracted from a variety of tumours2, a logical step would be to determine the subcellular localization of the specific antigen(s) on or within the cancer cell and whether or not the antigen continues to be associated with human malignant (or transformed) cells long maintained in culture. If this were so, then such cultures might serve as a convenient and standard source for an antigen of considerable potential in the diagnosis—and perhaps also the treatment—of malignant disease. The allied question of the identity and uniqueness of the antigens extractable from different cell lines or tumours will be the subject of a separate communication.