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.

Identification of molecular species of glycerophospholipids and sphingomyelin using electrospray mass spectrometry.

This paper describes the use of positive and negative ion electrospray mass spectrometry (MS) and MS/MS (tandem mass spectrometry) to identify glycerophospholipid and ceramide headgroups and their alkyl, alkenyl and acyl constituents. Molecular ion adducts were the primary products formed by positive ionization, occurring as [M+H]+, [M+Na]+, [M+K]+, [M+formate]+, or [M+acetate]+, depending upon the class of glycerophospholipid and the presence or absence of these ionization-promoting species. Similar (negatively charged) ions corresponding to the loss of the groups listed above were formed in negative ion MS. Positive ion electrospray MS/MS provides information on the nature of the headgroup, with the formation of an ion corresponding to the headgroup itself, or the loss of the headgroup from the molecular ion H+ or Na+ adduct. Acyl constituents are identified during negative ion MS/MS from the formation of their RCOO- ions. The nature of alkyl or alkenyl substituents in glycerophosphoethanolamine (PE) molecular species can be identified from residual ions following the loss of ethanolamine plus loss of the acyl moiety in the sn-2 position, and cyclization of a phosphate oxygen with C-2 of glycerol. In glycerophosphoinositol (PI) species, it appears that an RCO- ion is formed during negative ion MS/MS, possibly to steric interference from the bulky phosphoinositol headgroup that prevents cyclization (and subsequent stabilization) of the ion described for PE species. Positive and negative ion electrospray MS spectra for molecular species of commercial preparations of PE, PI, phosphatidylserine (PS), glycerophosphocholine (PC) and sphingomyelin (SM) produced similar profiles. For phospholipids occurring as Na+ adducts, concentrations above ca. 1 ng/microliter produced significant quantities of both [M+H]+ and [M+Na]+ ions for those molecular species present in the largest quantities, complicating interpretation of the spectra. Complete profiles of molecular species were obtained from as little as 10 picograms of material. Major components of PE were identified from 0.1 picogram total lipid. Using single ion monitoring of the Na+ adduct of beta-acetyl-gamma-O-hexadecyl L-alpha-phosphatidylcholine, 10 femtograms of material was detected. A mixture of 1 nanogram each of PE, PI, PS, and PC was readily resolved into individual molecular species, with little apparent loss of resolution or preferential ionization. Electrospray MS did not provide information on the position (sn-1 or sn-2) of fatty acids, and was not capable of differentiating in all instances between alkyl-acyl and alkenyl-acyl substituents without prior separation of these lipid subclasses.(ABSTRACT TRUNCATED AT 400 WORDS)

Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation.

Phosphatidylserine (PS) is asymmetrically distributed in mammalian cell membranes, being preferentially localized in the inner leaflet. Some studies have suggested that a disturbance in the normal asymmetric distribution of PS--e.g., PS exposure in the outer leaflet of the cell membrane, which can occur upon platelet activation as well as in certain pathologic red cells--serves as a potent procoagulant surface and as a signal for triggering their recognition by macrophages. These studies suggest that the regulation of PS distribution in cell membranes may be critical in controlling coagulation and in determining the survival of pathologic cells in the circulation. In this paper we describe a sensitive technique, based on PS-dependent prothrombinase complex activity, for assessing the amount of PS on the external leaflet of intact viable cells. Our results indicate that tumorigenic, undifferentiated murine erythroleukemic cells express 7- to 8-fold more PS in their outer leaflet than do their differentiated, nontumorigenic counterparts. Increased expression of PS in the tumorigenic cells directly correlated with their ability to be recognized and bound by macrophages.