A Study on Uptake and Localization of Merocyanine 540 (MC540) in Murine Myeloid Leukemia Cells by Confocal Laser Scanning Microscopy (CLSM)

The intracellular localization of merocyanine 540 (MC540), a photosensitizer commonly used in the photo-inactivation of leukemia cells, was studied using confocal laser scanning microscopy. It was found for the first time that MC540 not only localized in the plasma membrane but also in the cytoplasm and the nuclear membrane of the murine myeloid leukemia M1 and WEHI 3B (JCS) cells. Exposure of MC540 treated leukemia cells to light under conditions that could cause photobleaching did not cause the redistribution of cell-bound MC540. Rapid localization of MC540 in the cytoplasm was observed 5 minutes after exposure of leukemia cell to MC540, indicating that MC540 could promptly be internalized by these two leukemia cell lines. In contrast, localization of MC540 was limited only to the plasma membrane of erythrocytes. These results suggest that the binding pattern of MC540 is cell type dependent and may be related to the efficacy of photosenitization in photodynamic therapy.     

Hemocytes ultrastructure of Aedes aegypti (Diptera: Culicidae)

Mosquitoes have an efficient defence system against infection. Insect blood cells (hemocytes) play an essential role in defense against parasites and other pathogenic organisms that infect insects. We have identified by light and transmission electron microscopy six hemocytes cell types from the hemolymph of Aedes aegypti. They were: prohemocytes (20%), adipohemocytes (29%), granulocytes (16%), plasmatocytes (27%), oenocytoids (7%) and thrombocytoids (0.9%). The prohemocytes were the smallest hemocytes found in the hemolymph. Its cytoplasm occupies only a narrow area around the nucleus. The adipohemocytes were the most abundant cell type presented. These hemocytes exhibited a large lipid like vesicle and mitochondria. In electron micrographs, the granulocytes showed cytoplasm containing dilated rough endoplasmic reticulum (RER) and a round or elongated mitochondria. Electron-dense granules with a proteinaceous material were also present. The plasmatocytes were polymorphic and exhibited plasma membrane with irregular processes, philopodia and pseudopodia. Ultrastructural investigation revealed that the reticular cytoplasm showed a well-developed RER, a Golgi and vacuoles. Oenocytoids showed homogeneous cytoplasm with many mitochondria and ribosomes are scattered throughout the cytoplasm, abundant RER and a small smooth endoplasmic reticulum (SER) present at the cell poles. Thrombocytoids were very fragile and few in number. Similar characteristics were found in oenocytoids, possessing a homogeneous cytoplasm with poorly developed organelles, few mitochondria and granules.     

Histology and ultrastructure of pericardial cells of Scaptotrigona postica Latreille (Hymenoptera, Apidae) in workers and queens of different ages

The paper presents a study of the pericardial cells of Scaptotrigona postica an eusocial Brazilian stingless bee. Light and electron microscopy was used in a comparative study on workers and queens of different ages, exerting different functions in the colony. The pericardial cells are found only in the pericardial sinus, mainly in groups around the dorsal vessel. Each cell is enclosed by the basal membrane and its peripheral region is characterized by folds of the plasma membrane, which form canals and loops. The points where the plasma membrane folds is frequently closed by diaphragms, that along with the basal lamina form a barrier to substances from hemolymph. Along the membrane limiting the canals and loops, an intense endocytic activity through coated vesicles takes place indicating a selective absorption of hemolymph components. In older individuals, workers or queens, the cells exhibit larger quantities of cytoplasm inclusions, heterogeneous vacuoles containing the final products of intracellular digestion, and autophagic vacuoles with concentric membranous structures. The pericardial cells general morphology is in accordance with the role in processing metabolites captured from hemolymph and storage of indigested residues.     

Fluorescence quenching of fluorescein by Merocyanine 540 in liposomes

The fluorescence quenching of fluorescein (FL) by merociyanine 540 (MC540) was examined in L-egg lecithin phosphatidycholine (PC) liposomes using spectroscopic methods. The type of quenching mechanism (dynamic or static) was evaluated using the Stern-Volmer plots. Findings were also supported by the temperature studies and florescence decay measurements. The Stern-Volmer equation was utilized to calculate bimolecular quenching constants (K_q). Furthermore, the bimolecular quenching constant of the quencher in the liposomes (K_SV), partition coefficient (K_p), binding constant (K), and corresponding thermodynamic parameters ΔH, ΔS, and ΔG were calculated. The quenching property was also used in determining quantitatively (K_p) the partition coefficient of Merociyanini 540 in PC liposome.The obtained data indicated that static quenching occurred in the system and the K_SV values decreased with increasing lipid concentration. In addition, thermodynamic analysis suggested that van der Waals interactions and hydrogen bonding were the main acting forces between fluorescein and merociyanine 540 molecules in the medium.     

Distribution of merocyanine 540 in phospholipid membranes

The interaction of the fluorescent photosensitizer merocyanine 540 (MC-540) with model phospholipid membranes was studied. Two different-colored species (monomers and dimers) of MC-540 were registered in phospholipid liposomes. Variations in both phospholipid composition (DMPC, DPPC, POPC, egg PC) and temperature (15–60°C) resulted in changes in the MC-540 monomerdimer distribution. The values of the monomer-dimer equilibrium constant of MC-540 in egg PC (K=14.8 M), in POPC (K=26.7 M), and in DMPC (K=271.0 M) were determined at the temperature of 23±2°C. Suppression of MC-540 association with phospholipid bilayers was provoked by the addition of albumin to a liposome suspension. Albumin was observed to compete very successfully with lecithins containing saturated fatty acid chains (DPPC, DMPC), while only a weak competition of albumin with unsaturated lecithins (POPC, egg PC) for binding MC-540 molecules was registered.     

Ultrastructural localization of the RNA of immunodeficiency viruses using electron microscopy in situ hybridization and in vitroinfected lymphocytes

Cells infected in vitro with immunodeficiency viruses have been examined by electron microscopy in situ hybridization (EM ISH) methods for localization of viral RNA. Techniques used for preparation of specimens and probes are described. Unambiguous positive results were obtained using a mixture of two or three single negative strand DNA oligonucleotides complementary to regions of the gag, env and nef genes, each 200-300 bases and labelled with dig-11-UTP. Positive strand probes were used as a negative control. Cells were fixed with a mixture of formaldehyde and glutaraldehyde, dehydrated in ethanol with progressive lowering of temperature and embedded in Lowicryl K4M or HM20 at -35 degrees C. Permeabilization or pre-treatment of sections with proteinase K was not essential. The hybridization mixture was applied for 3-4h at 37 degrees C and probe was visualized by direct immuno-staining with sheep anti-digoxigenin antibodies conjugated to 10nm gold. This method would be suitable for future studies of the pathogenesis of retroviral infections and as a basis for further development of the EM ISH technique. EM ISH of in vitro infections of immunodeficiency viruses has shown the location of viral RNA in immature and mature viruses and its relationship to multimerized Gag protein during viral budding. The label for RNA has also been found in the cytoplasm of infected cells; it was mainly located adjacent to the plasma membrane and unassociated with visible Gag proteins. This may indicate that viral RNA migrates to the plasma membrane independently of the Gag protein and may, in some instances, arrive at the plasma membrane prior to the Gag protein. Viral RNA has also been found in the nucleus of peripheral blood mononuclear cells (PBMC) that were showing no morphological evidence of infection. The RNA was typically located in the nucleolus and in peripheral dense chromatin. These cells, which displayed morphological features of macrophage lineage, may have been the initial cell type to be infected in the PBMC.     

Ultrastructural immunogold localization of major sperm protein (MSP) in spermatogenic cells of the nematode Acrobeles complexus (Nematoda,Rhabditida)

The nematode spermatozoa represent a highly modified (aberrant) type of male gametes that lack a flagellum but for which the process of spermatogenesis culminates in the production of a crawling spermatozoon on the basis of the cytoskeletal component known as “major sperm protein”, or MSP. MSP is also known as an important hormone triggering oocyte maturation and ovulation in the model nematode Caenorhabditis elegans, where this protein was first identified. However, direct evidence of MSP localization and of its fate in nematode spermatogenic cells is rare. In this study, the spermatogenesis and sperm structure in the rhabditid nematode Acrobeles complexus (Rhabditida: Tylenchina: Cephalobomorpha: Cephaloboidea: Cephalobidae) has been examined with electron microscopy. Morphological observations were followed by high-pressure freezing and freeze-substitution fixation which allows post-embedding immunogold localization of MSP in all stages of sperm development using antibodies raised for MSP of C. elegans. In spermatocytes, synthetic activity results in the development of specific cellular components, fibrous bodies (FB) and membranous organelles (MO), which appear as FB-MO complexes where the filamentous matter of FB has been MSP-labeled. The spermatids subdivide into a residual body with superfluous cytoplasm, and a main cell body which contains nucleus, mitochondria and FB-MO complexes. These complexes dissociate into individual components, MO and FB, with the MSP being localized in FB. Immature spermatozoa from testes are opaque cells where a centrally located nucleus is surrounded by mitochondria, MO and FB clustered together, the MSP still being localized only in FB. Cytoplasm of mature spermatozoa from spermatheca is segregated into external pseudopods lacking organelles and a central cluster of mitochondria with intact MO surrounding the central nucleus. The FB ultimately disappear, and the MSP labeling becomes concentrated in the filamentous content of pseudopods and cytoplasm of the main cell body. Although the spermatogenesis and sperm structure of A. complexus is similar to that of many other rhabditid nematodes, their intact MO makes the morphology of the mature spermatozoa distinct from the “rhabditid pattern” and may be considered as a synapomorphy. The MSP localization in spermatogenic cells of A. complexus also follows the “rhabditid pattern” described in C. elegans and Ascaris spp. Our results and techniques of MSP labeling of A. complexus spermatogeneous cells reveal new possibilities to elucidate different research questions on MSP localization in nematodes related to C. elegans. Furthermore, the laboratory-cultured A. complexus strains can be used as a new and fascinating model to study MO and MSP functions in nematode reproduction.     

Relative quantification of cellular sections with molecular depth profiling ToF-SIMS imaging

We report the use of secondary ion mass spectrometry (SIMS) imaging to quantify the relative difference in the amount of lipid between two sections, the plasma membrane and the cytoplasm, of single cells from two different populations. Cells were each labeled with lipophillic dyes, frozen, fractured and analyzed in a ToF-SIMS mass spectrometer equipped with a 40 keV C₆₀⁺ ion source. In addition to identifying cells from separate populations, the lipophilic dyes can be used as a marker for the outer leaflet of the cell membrane and therefore as a depth finder. Here, we show that it is possible to compare the amount of lipids with particular headgroups in the cell membrane of a treated cell to the membrane of a control cell. Following erosion of the cell membranes, the amount of the two specific lipid head groups in the cytoplasm of the treated cell can be compared to those lipids in a control cell. Here we take the first step in this experimental design and display the ability to analyze multiple sections of frozen cells following a single fracture.     

Selective photoreceptor damage in four species of insects induced by experimental exposures to UV-irradiation

Damage to photoreceptive cells of insect compound eyes exposed to abnormally high doses of UV-radiation of 350nm peak wavelength manifests itself in at least two different ways. In the butterflies Papilio xuthus and Pieris napi from Japan and northern Finland, respectively, only the cell bodies of retinula cells 1 and 2, (identified as short wavelength receptors), but not their corresponding rhabdomeres, exhibit damage with apoptotic features. In the eye of UV-irradiated adult crickets, however, cell bodies and cytoplasm remain normal, while the rhabdomeres of cells 7 and 8 exhibit signs of severe membrane disruptions. No signs of damage whatsoever occurred in the eyes of northern Finnish bumblebees exposed to UV. It is suggested that metabolic shortfalls in the UV-sensitive cells of the butterfly eyes result in cellular shut-down, but that in the cricket receptors UV-induced changes of the membrane lipids dominate, leading to membrane instability without concomittant cell death. The strong resistance of the bumblebee eye to UV-induced damage requires further investigation, but since preconditioning to light can reduce photic damage in the rat eye, the 24h daylight experienced by northern Finnish bumblebees during the summer season could be involved.     

Freeze-thawing single human embryonic stem cells induce e-cadherin and actin filament network disruption via g13 signaling

Poor adhesion of single human embryonic stem (hES) cells after freeze-thawing causes death. To investigate mechanisms responsible for this, Rho-dependent protein kinase (ROCK) inhibitor Y-27632-treated and untreated single hES cells were analyzed for E-cadherin and F-actin distribution by immunostaining and phalloidin staining respectively and for G13 signaling pathway components by DNA microarray and quantitative polymerase chain reaction (PCR). Y-27632-treated cells clustered rapidly and maintained E-cadherin and F-actin distribution without losing Oct-3/4. Immediately after thawing, E-cadherin in untreated hES cells dotted along the membrane and then displayed eccentric cytoplasmic localization. Bleb formation and early Oct-3/4 loss occurred after F-actin network condensation in the cytoplasm. Microarray analyses and quantitative PCR indicated upregulation of two actin reorganization-associated components of the G13 signaling pathway, Arhgdib and Cdc42, in untreated cells. Considering these findings and that cell death was partly interrupted by Y-27632, E-cadherin and actin cytoskeleton network disruption through the G13 signaling pathway may cause hES cell death after freeze-thawing.     

Characterization of bioactive RGD peptide immobilized onto poly(acrylic acid) thin films by plasma polymerization

Plasma surface modification can be used to improve the surface properties of commercial pure Ti by creating functional groups to produce bioactive materials with different surface topography. In this study, a titanium surface was modified with acrylic acid (AA) using a plasma treatment and immobilized with bioactive arginine-glycine-aspartic acid (RGD) peptide, which may accelerate the tissue integration of bone implants. Both terminals containing the -NH₂ of RGD peptide sequence and -COOH of poly(acrylic acid) (PAA) thin film were combined with a covalent bond in the presence of 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC). The chemical structure and morphology of AA film and RGD immobilized surface were investigated by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). All chemical analysis showed full coverage of the Ti substrate with the PAA thin film containing COOH groups and the RGD peptide. The MC3T3-E1 cells were cultured on each specimen, and the cell alkaline phosphatase (ALP) activity were examined. The surface-immobilized RGD peptide has a significantly increased the ALP activity of MC3T3-E1 cells. These results suggest that the RGD peptide immobilization on the titanium surface has an effect on osteoblastic differentiation of MC3T3-E1 cells and potential use in osteo-conductive bone implants.     

Caveolae and caveolin isoforms in rat peritoneal macrophages

Caveolea are special (highly hydrophobic) plasma membrane invaginations with a diameter of 50-100 nm. Their characteristic features are the flask- or omega-shape and the lack of basket-like coat composed of clathrin. Caveolin-an integral membrane protein-is the principal component of caveolae membranes in vivo. Multiple forms of caveolin have been identified: caveolin-1alpha, caveolin-1beta, caveolin-2 and caveolin-3. They differ in their specific properties and tissue distribution. In this paper we summarize the morphological and biochemical data providing strong evidence about the existence and function of caveolae in rat peritoneal macrophages. When studied electron microscopically, the surface of both resident and elicited macrophages exhibited omega- or flask-shaped plasma membrane invaginations. There was a significant difference, however, in the number of these profiles: whereas in resident cells only a small amount of them was found on the cell surface, in elicited cells they were abundantly present on the plasma membrane. Using an antibody against the VIP21/caveolin-1 isoform we showed that these plasma membrane pits were indeed caveolae. The number and the appearance of caveolae were found to be in close correlation with the functional activity of these phagocytotic cells, indicating that the formation of caveolae is a highly regulated process.Using Western blot analysis two different proteins ( approximately 29 and approximately 20 kDa)-both labelled with anti-caveolin antibodies-were identified in resident and elicited macrophages that have been isolated from rat peritoneal cavity. The approximately 20 kDa protein was labelled specifically only by anti-VIP21/caveolin-1, while the approximately 29 kDa protein was labelled by both anti-VIP21/caveolin-1 and anti-caveolin-2 antibodies. The presence of the approximately 29 kDa protein was highly characteristic of resident cells, and only a small amount of approximately 20 kDa protein was detected in these cells. Elicitation has resulted in a significant increase in the amount of approximately 20 kDa protein labeled only with anit-VIP21/caveolin-1. Our morphological (confocal and electron microscopical) studies have shown that in resident cells caveolin was present in the cytoplasm, in smaller vesicles and multivesicular bodies around the Golgi area. Only a very small amount of caveolae was found on the cell surface of these cells. In elicited macrophages, caveolae (labelled with anti-VIP21/caveolin-1 antibody) appeared in large numbers on the cell surface, but caveolin detected by anti-caveolin-2 was also found in small vesicles and multivesicular bodies. These data support the idea that the expression of the approximately 29 kDa (caveolin-related) protein is insufficient for caveolae formation in resident cells, it can function as a modified, macrophage-specific caveolin-2 isoform.Our results strongly suggest that caveolin-1 plays a crucial role in the formation of caveolae: it is the amount of caveolin-1 that regulates the appearance of caveolae on the plasma membrane.Studying the endocytotic processes of resident and elicited macrophages we have found that elicited macrophages bound and internalized significantly larger amounts of fluid phase marker (HRP) and immune complex (peroxidase-antiperoxidase-PAP) than resident cells. Serial section analysis, double labelled immunocytochemistry, and filipin treatment were used to demonstrate that caveolae can pinch off from the plasma membrane and can take part in endocytotic processes as alternative carriers in elicited macrophages.     

Structural Changes in Lymphocytes Membrane of Chernobyl Clean-up Workers from Latvia

ABM (3-aminobenzanthrrone derivative) developed at the Riga Technical University, Riga, Latvia) has been previously shown as a potential probe for determination of the immune state of patients with different pathologies .The fist study (using probe ABM) of peripheral blood mononuclear cells (PBMC) membranes of 97 Chernobyl clean-up workers from Latvia was conducted in 1997. Now we repeatedly examine the same (n = 54) individuals in dynamics. ABM spectral parameters in PBMC suspension, fluorescence anisotropy and blood plasma albumin characteristics were recorded. In 1997 screening showed 5 different patterns of fluorescence spectra, from which in 2007 we obtained only two. These patterns of spectra had never been previously seen in healthy individuals or patients with tuberculosis, multiple sclerosis, rheumatoid arthritis, etc., examined by us. Patterns of ABM fluorescence spectra are associated with membrane anisotropy and conformational changes of blood plasma albumin. We observed that in dynamics 1997–2007 the lipid compartment of the membrane became more fluid while the lipid-protein interface became more rigid. The use of probe ANS and albumin auto-fluorescence allowed show conformational alterations in Chernobyl clean-up workers blood plasma. It is necessary to note that all investigated parameters significantly differ in observed groups of patients. These findings reinforce our understanding that that the cell membrane is a significant biological target of radiation. The role of the membrane in the expression and course of cell damage after radiation exposure must be considered. So ten years dynamic of PBMC membrane characteristics by ABM (spectral shift and anisotropy indexes) in Chernobyl clean-up workers reveal progressive trend toward certain resemblance with those of chronic B-cell lymphoid leukemia.     

Fluorescence Correlation Spectroscopy (FCS) Analysis of Human Red Blood Cell System

Recent studies have revealed the importance of the lipid micro domain for signal transduction in cell membrane. To analyze the biophysical properties of the lipid micro domain at the single molecule level, we measured the diffusion of fluorescence probe in human red blood cell (RBC) membrane using fluorescence correlation spectroscopy (FCS). The value of diffusion constant of octadecyl rhodamine B chloride (R18), D = 4.7 × 10⁻⁹cm²/s, is close to that of phospholipid molecules in membrane. This indicates that the probed RBC with R18 could be a convenient model for analyzing membrane property under natural conditions. Using this model, we investigated how amyloid beta peptide (A-beta) interacts with plasma membrane. This paper demonstrates that A-beta was inserted into the phospholipid bilayer of the RBC membrane and predicts the existence of the micro domain, lipid raft, on this membrane by the heterologous insertion of A-beta.     

Ultrastructural element localization by EDXS in Empetrum nigrum

Empetrum nigrum L. is one of the few species growing on highly polluted areas in the northern boreal forests and it accumulates considerable amounts of heavy metals especially in its older stems. Previous-year stems of Empetrum nigrum were collected from two different sites located at distances of 0.5km (highly contaminated) and 8km (low contaminated) from a Cu--Ni smelter at Harjavalta, SW Finland. The element (Al, As, Cu, Fe, Mn, Zn, Ca, K, P, S, Mg, Na) localization was performed by energy-dispersive X-ray spectroscopy (EDXS) after cryofixation, freeze-drying and pressure infiltration of the material. The results showed higher levels of Cu, As and Fe in cell compartments of E. nigrum close to the smelter than at further distance. The Al and Zn levels, in contrast, showed no clear differences between the sites. Cu was distributed homogeneously in the tissue and occurred in vacuoles, cytoplasm, cell walls as well as in lumens of the vascular tissue. The higher amounts of As were localized in the outer regions of the stem cross-section and the amounts were higher in the primary cell walls of living (ray cells, phloem) than dead cells (xylem, sclereids). Ray cells, phloem and sclereids had elevated Fe amounts compared to the other tissues in the contaminated stem samples but owing to the high variation between the replicates, no significant differences were found. Based on the rather homogeneous localization of Cu, As and Fe in the living tissue and increased levels of Cu, As and Fe in vacuoles, cell walls and cytoplasm near the smelter, it seems that more than one specific mechanism contribute to the heavy metal tolerance of E. nigrum. Macronutrients did not show clear differences between the two distances or connection to heavy metal localization. Neither the role of complexing agents in heavy metal tolerance in the cytoplasm or vacuoles could be shown by this study. Because of the more frequent localization of electron dense phenolic material in the polluted samples, it might also have a function in the heavy metal tolerance of E. nigrum.     

Ternary fission induced by 540 MeV Fe ions on uranium

The ratio of ternary to binary fission for uranium irradiated with 540 MeV Fe ions was found to be (4.3 ±0.7)%, when fission fragments in the forward hemisphere of the laboratory system are investigated. The detector was mica, heated after irradiation for 1 hour at 500°C, so as to reveal only fission fragments as tracks. The relatively high yield of ternary fission is interpreted qualitatively on the basis of potential-energy curves for binary fission and prolate ternary fission. The two curves appear to be fairly similar for the two decay modes.     

Plasma surface modification of nanofiltration (NF) thin-film composite (TFC) membranes to improve anti organic fouling

Commercial nanofiltration (NF) thin-film composite (TFC) membranes were treated by low-pressure NH₃ plasma, and the effects of the plasma treatment were investigated in terms of the membrane hydrophilicity, pure water flux, salt rejection, protein adsorption, and humic acid fouling. Experimental results indicated that the membrane surface hydrophilicity was increased by the plasma treatment, and changes in the hydrophilicity as well as membrane performance including permeate flux and fouling varied with the original membrane characteristics (e.g., roughness and hydrophilicity). Water flux of plasma treated membranes was the highest with 10 min and 90 W of plasma treatment, and salt rejection was mainly affected by the intensity of the plasma power. Results of bovine serum albumin (BSA) adsorption demonstrated that the protein adsorption decreased with increasing plasma treatment time. The plasma treatment that resulted in more negatively charged surfaces could also better prevent Aldrich humic acid (AHA) attachment on the membrane surface.     

采用氧合血红蛋白吸收带的漫反射光谱比率R540/R575检测人胃癌

采用氧合血红蛋白吸收带的平均漫反射光谱比率(R540/R575)对离体的人正常胃、未分化胃腺癌、胃鳞状细胞癌和低分化胃腺癌的上皮粘膜组织进行鉴别诊断.实验采用带积分球附件的分光光度计测量组织样品在400 nm～600 nm的漫反射光谱.实验结果表明,在相同的波长范围内,人的未分化胃腺癌、胃鳞状细胞癌和低分化胃腺癌的上皮粘膜组织的漫反射光的强度较人正常胃的上皮粘膜组织的漫反射光强度要显著地低的,尤其是在氧合血红蛋白的吸收带540 nm和575 nm波长处,人正常胃、未分化胃腺癌、胃鳞状细胞癌和低分化胃腺癌的上皮粘膜组织的平均漫反射光谱比率分别是(94.25±2.4)%,(109±3.0)%,(1032±2.8)%和(98.6±2.6)%.     

Ultrasound exposure in the presence of hematoporphyrin induced loss of membrane integral proteins and inactivity of cell proliferation associated enzymes in sarcoma 180 cells in vitro

Ultrasonically induced effects of hematoporphyrin (HPD) on cell damage and membrane protein alteration of S180 isolated tumor cells in vitro were investigated, and the potential mechanisms of sonodynamic therapy (SDT) inhibiting tumor growth were discussed. Tumor cells suspended in air-saturated PBS (pH 7.2) were exposed to ultrasound at 1.8 MHz for up to 180 s in the presence and absence of HPD. The viability of cells was determined by a trypan blue exclusion test. To estimate the damage effects of SDT on plasma membrane of tumor cells primarily, membrane integral proteins (EGFR, Ras, Fas, FasL) and cell proliferation associated enzymes (adenylate cyclase and guanylate cyclase) were checked with immunochemical methods. The results indicated that the intensity threshold for ultrasonically induced cell damage at 1.8 MHz was 3 W/cm². At this condition, the expression of the integral proteins was obviously inhibited and the activity of the enzymes was decreased post ultrasound treatment in the presence of 20 μg/ml HPD. Loss of the membrane proteins and inactivity of AC and GC post SDT was time-dependent. This paper reveals SDT can cause the loss of tumor cell membrane integral proteins and inactivity of the enzymes associated with cell proliferation which might be attributed to a sonochemical activation mechanism. The mechanisms by that tumor growth is inhibited by SDT can be understood as that the growth signaling pathway is partially interdicted and the resistance of tumor cells to the specifically activated immune cells is weakened.     

Sonodynamic effects of protoporphyrin IX disodium salt on Ehrlich ascetic tumor cells

The cytotoxic effect of protoporphyrin IX disodium salt (PPIX) on isolated Ehrlich ascetic tumor (EAT) cells induced by ultrasound exposure was investigated. Tumor cells suspended in air-saturated phosphate buffer solution (PBS, pH 7.2) were exposed to ultrasound at 2.2 MHz for up to 60 s in the presence and absence of PPIX. The viability of cells was determined by a trypan blue exclusion test. The morphological changes of cells in SDT were observed by scanning electron microscope (SEM). And the sub-cellular localization of PPIX in EAT cells was detected by confocal laser scanning microscopy (CLSM). The ultrasonically-induced cell damage increased as PPIX concentration increased, while no cell damage was observed with PPIX alone. CLSM observation revealed that the fluorescence of PPIX and rhodamine 123 (mitochondrial probe) overlapped very well in the cytoplasm. The results indicate that PPIX could enhance the ultrasonically-induced cell damage and mitochondria may play an important role during sonodynamically induced cytotoxicity.