P-glycoprotein subcellular localization and cell morphotype in MDR1 gene-transfected human osteosarcoma cells.

Efflux of chemotherapy agents by P-glycoprotein at the plasma membrane is thought to be a major cause of cancer multidrug-resistance (MDR). However, the mechanism underlying the cellular accumulation and distribution of cytotoxic drugs is still poorly defined. We have recently found that P-glycoprotein is expressed also in the nucleus of MDR cell lines selected in doxorubicin (DXR), suggesting the possible involvement of this protein in the direct extrusion of the drug from the nucleus of resistant cells. In this study, we analyzed the subcellular localization of P-glycoprotein, in a series of U-2 OS osteosarcoma cell clones transfected with MDR1 gene in order to verify whether the nucleus is a constant site for the localization and functional activity of P-glycoprotein, and in which way some aspects of cell morphology related to MDR depend on the subcellular P-glycoprotein localization rather than on the exposure to the selective drug. Our results indicate that to achieve a subcellular drug distribution prevailing in the cytoplasm but not in the nucleus, a significant increase in the expression of P-glycoprotein at the different cellular compartments, including the plasma membrane, the cytoplasm, and the nucleus, is needed, although the in vitro drug resistance appears to be mainly dependent on the expression of P-glycoprotein at the cell surface. With regard to the morphological characteristics of MDR cells involving the cell surface and the chromatin arrangement, the influence of DXR appears to be prevalent, although P-glycoprotein overexpression cannot be excluded.     

Light and electron microscopy and molecular phylogenetic analyses of Chloromonas pseudoplatyrhyncha (Volvocales,Chlorophyceae)

A strain of Chloromonas pseudoplatyrhyncha (Pascher) P. C. Silva, which has not been studied previously using cultured material, was established from a soil sample collected in Japan and examined by light microscopy, transmission electron microscopy, and molecular phylogenetic analyses. The chloroplasts of this species showed no pyrenoids under light microscopy. However, transmission electron microscopy and the staining methods with carmine after fixation in an acidified hypochlorite solution revealed that Chloromonas pseudoplatyrhyncha actually had multiple, atypical pyrenoids (pyrenoid matrices without associated starch grains) that were angular in shape and distributed in the interior regions of the lobes of the chloroplasts. Although some other species of Chloromonas have atypical pyrenoids in the chloroplast, such angular pyrenoids have not previously been reported within the Volvocales. The present molecular phylogenetic analysis, based on 18S ribosomal RNA, adenosine triphosphate synthase β‐subunit, and P700 chlorophyll a‐apoprotein A2 gene sequences, demonstrated that Chloromonas pseudoplatyrhyncha belonged to the Chloromonas lineage or Chloromonadinia, in which it occupied a basal position outside a robust, large monophyletic group consisting of 13 species of Chloromonas and Gloeomonas.     

Researching into the cellular shape, volume and elasticity of mesenchymal stem cells, osteoblasts and osteosarcoma cells by atomic force microscopy

Within the bone lie several different cell types, including osteoblasts (OBs) and mesenchymal stem cells (MSCs). The MSCs are ideal targets for regenerative medicine of bone due to their differentiation potential towards OBs. Human MSCs exhibit two distinct morphologies: rapidly self-renewing cells (RS) and flat cells (FC) with very low proliferation rates. Another cell type found in pathological bone conditions is osteosarcoma. In this study, we compared the topographic and morphometric features of RS and FC cells, human OBs and MG63 osteosarcoma cells by atomic force microscopy (AFM). The results demonstrated clear differences: FC and hOB cells showed similar ruffled topography, whereas RS and MG63 cells exhibited smoother surfaces. Furthermore, we investigated how selected substrates influence cell morphometry. We found that RS and MG63 cells were flatter on fibrous substrates such as polystyrene and collagen I, but much more rounded on glass, the smoothest surface. In contrast, cells with large area, namely FC and hOB cells, did not exhibit pronounced changes in flatness with regards to the different substrates. They were, however, remarkably flatter in comparison to RS and MG63 cells. We could explain the differences in flatness by the extent of adhesion. Indeed, FC and hOB cells showed much higher content of focal adhesions. Finally, we used the AFM to determine the cellular Young's modulus. RS, FC and hOB cells showed comparable stiffness on the three different substrates, while MG63 cells demonstrated the unique feature of increased elasticity on collagen I. In summary, our results show, for the first time, a direct comparison between the morphometric and biophysical features of different human cell types derived from normal and pathological bone. Our study manifests the opinion that along with RNA, proteomic and functional research, morphological and biomechanical characterization of cells also reveals novel cell features and interrelationships.     

miR-17-5p promotes proliferation and epithelial-mesenchymal transition in human osteosarcoma cells by targeting SRC kinase signaling inhibitor 1

MicroRNA-17-5p (miR-17-5p) and epithelial-mesenchymal transition (EMT) have been reported to participate in the development and progression of multiple cancers. However, the relationship between the miR-17-5p and EMT in osteosarcoma (OS) is still poorly understood. This study was to investigate the effects of the miR-17-5p and its potential mechanism in regulating proliferation, apoptosis, and EMT of human OS. Quantitative real-time PCR was used to detect the miR-17-5p and SRC kinase signaling inhibitor 1 (SRCIN1) messenger RNA expression in OS specimens and cell lines. After transfection with miR-17-5p inhibitors, proliferation, apoptosis, migration, and invasion of OS cells were assessed by using the Cell Counting Kit-8, the annexin V-FITC apoptosis, wound-healing, and transwell assays. The SRCIN1 was validated as a target of the miR-17-5p through bioinformatics algorithms and luciferase reporter assay. Moreover, the expression of EMT markers, E-cadherin, N-cadherin, and Snail was identified by the Western blot analysis. MiR-17-5p was significantly upregulated in OS tumor samples and cell lines. It inhibited proliferation and EMT, and promoted apoptosis in OS. The SRCIN1 was identified as a direct target of the miR-17-5p. Silenced miR-17-5p could change the expression of EMT markers, such as upregulating the expression of E-cadherin, and downregulating the expression of N-cadherin and Snail through targeting the antioncogenic SRCIN1. These findings suggest that the miR-17-5p promotes cell proliferation, and EMT in human OS by directly targeting the SRCIN1, and reveal a branch of the miR-17-5p/SRCIN1/EMT signaling pathway involved in the progression of OS.     

Scanning electron microscopy of stromal cells of human placental villi throughout pregnancy

Summary Morphological changes in fixed stromal cells and Hofbauer cells were studied throughout pregnancy in different types of placental chorionic villi by scanning electron microscopy. In the mesenchymal villus the fixed stromal cells were characterized by thin cytoplasmic processes. Hofbauer cells exhibited blebs on their surface. Large sail-like processes with a crescent profile which surrounded well developed stromal channels and a small cell body typified the small reticulum cells of the immature intermediate villus. The Hofbauer cells here displayed blebs, microplicae and large lamellipodia. Short cytoplasmic expansions and a large cell body characterized the fibroblasts present inside the stem villus. Hofbauer cells were rare, having blebs or a few short lamellipodia. The mature intermediate villus contained small and large reticulum cells. The latter had a much larger cell body than the small ones and displayed a few short cytoplasmic processes partly delimiting narrow incomplete stromal channels. Occasional Hofbauer cells with small microplicae and/or blebs were present. The small reticulum cells and fibroblasts present in the terminal villus showed similar morphological features as above. However, the former exhibited less developed cytoplasmic extensions and therefore no stromal channels were observed. In the terminal villus, the morphology of the rare Hofbauer cells was similar to that found in the mature intermediate villus.     

Maturation of rat dendritic cells during intrahepatic translocation evaluated using monoclonal antibodies and electron microscopy

Specific populations of hepatic sinusoidal cells were stained with monoclonal antibodies that recognize monocytes/macrophages (ED1), tissue macrophages (Kupffer cells) (ED2), MHC class II (Ia) antigen (MRC OX6), and dendritic cells/γ,δ T-cells (MRC OX62) and analyzed by light and electron microscopy. The majority of ED1⁺ and/or ED2⁺ cells were localized to the hepatic parenchyma, whereas OX6⁺ and/or OX62⁺ cells were more densely distributed within Glisson’s sheath than in the hepatic parenchyma. Double-immunoperoxidase staining of normal liver for ED1, ED2, and OX6 identified dendritic cells (DC) of two different phenotypes, ED1⁺ED2^–OX6⁺ and ED1^–ED2^–OX6⁺. DC can be classified into three different types based on ultrastructural characteristics. The first type (type I) is characterized by one or more long cytoplasmic processes and a well-developed lysosomal system. The second type (type II) has an inconspicuous lysosomal system, abundant hyaloplasm, and characteristic short cytoplasmic processes. The third type (type I–II) has cytologic features intermediate between those of type I and type II DC. At the electron-microscopic level, these three cell types are found in the sinusoidal lumen, whereas the majority of type II DC are located in the space of Disse and Glisson’s sheath. Furthermore, some OX6-labeled elongated DC appeared to traverse the lumen of sinusoids through endothelial pores to enter the space of Disse. One hour after intravenous injection of latex particles (0.81 μm in diameter), numerous latex-laden dendritic cells (ED1⁺OX6⁺, type I and type I–II) were detected in the lumen of hepatic sinusoids, but not in the space of Disse or Glisson’s sheath. These findings suggest that normal rat liver contains resident dendritic cells which downregulate phagocytic activity and mature into potent accessory cells during migration from the portal vein toward the central vein. These DC then traverse the sinusoidal lumen to the hepatic lymph system via the space of Disse. Received: 8 May 1998 / Accepted: 15 June 1998     

Lethal effect of chitosan‐Ag (I) films on Staphylococcus aureus as evaluated by electron microscopy

Aim: This article investigated the lethal effect and morphological changes on Staphylococcus aureus strains ATCC 25923 and ATCC 6538P produced by chitosan‐Ag (I) films as observed by electron microscopy. Methods and Results: The antimicrobial activity of films against staphylococci was determined using the broth dilution method and agar diffusion test. Killing curves, transmission and scanning electron microscopy (TEM and SEM) techniques were employed to evaluate the bacterial death and morphological changes in bacterial cells after exposure to chitosan‐Ag (I) films. Films affected the cell structure of Staph. aureus, causing elongation of cells, disaggregation of grape‐like cluster, contraction of bacterial cytoplasm, thickening of cell wall, increase in cell wall roughness, cell disruption with loss of intracellular material, filamentation and bacteriolysis, as seen in the micrographs following 3, 6, 12 and 16 h of incubation. Conclusions: Obtained images clearly show that chitosan‐Ag (I) films have a notable antistaphylococcal activity. Significance and Impact of the Study: Information from this study can be employed in guiding future strategies to improve the design of materials for the food industry packaging.     

Blue LED causes autophagic cell death in human osteosarcoma by increasing ROS generation and dephosphorylating EGFR

Osteosarcoma (OS) is the most common primary malignant bone tumour in adolescence. Lately, light-emitting diodes (LED)-based therapy has emerged as a new promising approach for several diseases. However, it remains unknown in human OS. Here, we found that the blue LED irradiation significantly suppressed the proliferation, migration and invasion of human OS cells, while we observed blue LED irradiation increased ROS production through increased NADPH oxidase enzymes NOX2 and NOX4, as well as decreased Catalase (CAT) expression levels. Furthermore, we revealed blue LED irradiation-induced autophagy characterized by alterations in autophagy protein markers including Beclin-1, LC3-II/LC3-I and P62. Moreover, we demonstrated an enhanced autophagic flux. The blockage of autophagy displayed a remarkable attenuation of anti-tumour activities of blue LED irradiation. Next, ROS scavenger N-acetyl-L-cysteine (NAC) and NOX inhibitor diphenyleneiodonium (DPI) blocked suppression of OS cell growth, indicating that ROS accumulation might play an essential role in blue LED-induced autophagic OS cell death. Additionally, we observed blue LED irradiation decreased EGFR activation (phosphorylation), which in turn led to Beclin-1 release and subsequent autophagy activation in OS cells. Analysis of EGFR colocalization with Beclin-1 and EGFR-immunoprecipitation (IP) assay further revealed the decreased interaction of EGFR and Beclin-1 upon blue LED irradiation in OS cells. In addition, Beclin-1 down-regulation abolished the effects of blue LED irradiation on OS cells. Collectively, we concluded that blue LED irradiation exhibited anti-tumour effects on OS by triggering ROS and EGFR/Beclin-1-mediated autophagy signalling pathway, representing a potential approach for human OS treatment.     

Multiphoton tomography in differentiation of morphological and molecular subtypes of breast cancer: A quantitative analysis

In this study multiphoton tomography, based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) was used to visualize both the extracellular matrix and tumor cells in different morphological and molecular subtypes of human breast cancer. It was shown, that quantified assessment of the SHG based imaging data has great potential to reveal differences of collagen quantity, organization and uniformity in both low- and highly- aggressive invasive breast cancers. The values of quantity and uniformity of the collagen fibers distribution were significantly higher in low-aggressive breast cancer compared to the highly-aggressive subtypes, while the value representing collagen organization was lower in the former type. Additionally, it was shown, that TPEF detection of elastin fibers and amyloid protein may be used as a biomarker of detection the low-aggressive breast cancer subtype. Thus, TPEF/SHG imaging offers the possibility of becoming a useful tool for the rapid diagnosis of various subtypes of breast cancer during biopsy as well as for the intraoperative determinination of tumor-positive resection margins.     

Qualitative and quantitative preservation of the fine structure of absorptive cells in cultured biopsies of human small-intestine

Summary The fine structure of the absorptive cells in human small-intestinal biopsies cultured for 6, 24, and 48 h was analyzed qualitatively and quantitatively. The findings show generally good preservation of the cultured absorptive cells and a normal distribution, size, and relative volume of their cell organelles, but there was a systematic decrease in the apical cell surface and an increase in the number of apical vesicles and tubules after culturing. Since the apical vesicles and tubules are thought to have a function in the transport of cell-coat material from the Golgi apparatus to the cell surface, these findings raise the question of whether a delayed transport or extrusion of cell surface material occurs.The diminished relative volume of the mitochondria and the increased signs of autophagy in some poorly preserved absorptive cells, are assumed to be an adaption to less favourable culture conditions.The authors acknowledge the help of Dr. A.S. Pena in the setting up of the organ culture technique, and also wish to thank Mrs. M.L. Bouwhuis for statistical advice and Mrs. M. de Gruil and Mr. L.D.C. Verschragen for technical assistance. The investigations were supported in part by the Foundation for Medical Research (FUNGO), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO)     

粉尘螨生殖系统超微结构的透射电镜观察

本研究主要采用透射电镜观察粉尘螨Dermatophagoides farinae (Hughes)生殖系统超微结构。粉尘螨雄性生殖系统是由精巢、 输 精管、 附腺、 射精管、 交配器官及附属交配器官组成。精巢内可同时有精子发育各阶段的细胞。精子无核膜、 核染色质聚集成束、 线 粒体缺乏典型的嵴、 胞质内有平行排列的电子致密薄片等为其特征性结构。雌性生殖系统由交合囊、 交合囊管、 储精囊、 囊导管、 卵 巢、 输卵管、 子宫及产卵管构成。卵巢内可见含多个细胞核的中央细胞, 其周为卵母细胞等生殖细胞。该研究丰富了对粉尘螨生殖系统 结构的认识。     

Osteotoxicity of cadmium and lead in HOS TE 85 and ROS 17/2.8 cells: relation to metallothionein induction and mitochondrial binding

Epidemiological, experimental and clinical data indicate that cadmium and lead are osteotoxins in man and other species. The relative sensitivities of a clonal human osteosarcoma cell line (HOS TE 85) and a clonal rat osteosarcoma cell line (ROS 17.28) to the cytotoxic effects of cadmium and lead were tested in serum-free media without added growth factors. The rat osteosarcoma cells were more sensitive to cadmium with cytotoxicity and inhibition of proliferation at 0.25 versus 0.75 and 1.0 mol l^– cadmium, respectively, for human osteosarcoma cell lines. The lower sensitivity to cadmium of human osteosarcoma cells is attributed, at least partly, to induction of metallothionein synthesis by cadmium and zinc in this cell line; in the rat osteosarcoma cell line, they do not induce metallothionein synthesis. Human osteosarcoma cells were more sensitive than rat osteosarcoma cells to lead with inhibition (IC₅₀) of proliferation at 4 mol l^– lead and cytotoxicity at 20 versus 6 and over 20 moll^– lead, respectively, for these variables in rat osteosarcoma cells. Both cells lines attained the highest lead concentration in the 15 000 × g (mitochondrial) fraction. The lead in the mitochondrial, microsomal, nuclear and cytosolic fractions of the human cell line did not decrease during 24 h post-washout. Binding of lead was much less stable in the less sensitive rat cells, with 50–100% loss of mitochondrial, microsomal and nuclear lead during 24 h post-washout.     

Identification of quantitative trait loci for larval morphological traits in interspecific hybrids of Ochlerotatus triseriatus and Ochlerotatus hendersoni (Diptera: Culicidae)

Ochlerotatus triseriatus is the natural vector of La Crosse virus, a common cause of pediatric encephalitis in the United States; the closely related Ochlerotatus hendersoni transmits this virus at low frequency. Adults of these mosquito species are difficult to distinguish morphologically; however, the larval stages show species-specific differences in several characters. We identified genomic regions contributing to the differences between the larvae of these species through interspecific hybridizations. Quantitative trait loci (QTL) were identified by standard interval mapping techniques and by univariate marker association analyses. We examined 159 F₂ progeny from an Oc. hendersoni female by Oc. triseriatus male interspecific cross for variation in the number of saddle and siphon hair branches, attachment of the acus, and morphology of the anal papillae. At least one putative QTL was identified for each of the phenotypes examined. QTL most commonly mapped to linkage group (LG) III, although QTL were identified on LGI and LGII for three phenotypes each. Several of these QTL, and particularly those on LGIII, also map to genome regions controlling adult female body size and ability to orally transmit La Crosse virus. Further studies are required to elucidate the relationships among these traits and the impact they may have had on the ecological specialization and speciation of these mosquitoes.     

(Na-K)ATPase activity along the nephrons in normal and adrenalectomized rats measured by quantitative cytochemistry

A cytochemical method was used to measure total, ouabain insensitive and specific (Na-K)ATPase activities along the rat nephron. Enzyme activity was expressed as per cent of mean integrated extinction with reference to a calibrated filter. The lowest mean values of total, ouabain-insensitive, and (Na-K)ATPase activities were found in the proximal convoluted tubule (PCT). In the distal convoluted tubule (DCT), total and ouabain-insensitive activities (77.8 per cent and 45.8 per cent, respectively) were significantly higher than in the medullary thick ascending limb (MAL) (66.0 per cent and 24.6 per cent, respectively). Mean values of (Na-K)ATPase activity were significantly lower in DCT than in MAL (32.0 per cent and 41.3 per cent, respectively). Using Lineweaver-Burk plots, the KM ATP value for total ATPase activity was found to be 2.33, 1.79, and 3.63 mM in DCT, MAL, and PCT respectively. Maximal velocity was lower in PCT than in MAL and DCT. For (Na-K)ATPase, the smallest KM value was found in MAL (0.95 mM) and was 2.73 and 5.71 mM in DCT and PCT respectively. Maximal velocity was the highest in MAL (49.3 per cent), lower in DCT (36.1 per cent) and least in PCT (22.5 per cent). ATPase was measured in the MAL and DCT from rats fed a normal (N-Na+) or a high (Hi-Na+) sodium diet, and from Hi-Na+ rats one week after adrenalectomy (ADX). In the MAL, (Na-K)ATPase tended to be higher in Hi-Na+ than in rats, but was significantly lower in ADX than in Hi-Na+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of P-glycoprotein-mediated Multidrug Resistance (MDR) by N,N-bis(cyclohexanol)amine aryl esters: further restriction of molecular flexibility maintains high potency and efficacy

Conformational modulation of the aryl portion of a set of N,N-bis(cyclohexanol)amine aryl esters (1a-d) that are potent Pgp-dependent MDR inhibitors has been performed. Toward this end the trans-3-(3,4,5-trimethoxyphenyl)acrylic acid present in set 1 was substituted with 3-(3,4,5-trimethoxyphenyl)propanoic and 3-(3,4,5-trimethoxyphenyl)propiolic moieties to give sets 2 and 3, respectively. While the introduction of 3-(3,4,5-trimethoxyphenyl)propanoic moiety resulted in a definite drop in potency and efficacy, esterification with 3-(3,4,5-trimethoxyphenyl)propiolic acid gave four isomers (3a-d) that maintain high potency and possess optimal efficacy. These results are discussed in terms of conformational flexibility of the different sets of compounds.