The impact of TiO2 nanoparticles on uptake and toxicity of benzo(a)pyrene in the blue mussel (Mytilus edulis).

Nanoparticles are emerging contaminants of concern. Knowledge on their environmental impacts is scarce, especially on their interactive effects with other contaminants. In this study we investigated effects of titanium dioxide nanoparticles (TiO2NP) on the blue mussel (Mytilus edulis) and determined their influence on the bioavailability and toxicity of benzo(a)pyrene (B(a)P), a carcinogenic polyaromatic hydrocarbon (PAH). Blue mussels were exposed to either TiO2NP (0.2 and 2.0 mg L(-1)) or B(a)P (20 µg L(-1)) and to the respective combinations of these two compounds. Aqueous contaminant concentrations, the uptake of Ti and B(a)P into mussel soft tissue, effects on oxidative stress and chromosomal damage were analyzed. The uncoated TiO2NP agglomerated rapidly in the seawater. The presence of TiO2NP significantly reduced the bioavailability of B(a)P, shown by lowered B(a)P concentrations in exposure tanks and in mussel tissue. The activities of antioxidant enzyme superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were impacted by the various exposure regimes, indicating oxidative stress in the contaminant exposure groups. While SOD activity was increased only in the 0.2TiO2NP exposure group, CAT activity was enhanced in both combined exposure groups. The GPx activity was increased only in the groups exposed to the two single compounds. In hemocytes, increased chromosomal damage was detected in mussels exposed to the single compounds, which was further increased after exposure to the combination of compounds. In this study we show that the presence of TiO2NP in the exposure system reduced B(a)P uptake in blue mussels. However, since most biomarker responses did not decrease despite of the lower B(a)P uptake in combined exposures, the results suggest that TiO2NP can act as additional stressor, or potentially alters B(a)P toxicity by activation.

Overexpression of p150, a part of the large subunit of the eukaryotic translation initiation factor 3, in colon cancer.

BACKGROUND: P150, a 150 kDa protein, was isolated from virally and oncogene-transformed mouse cell lines, partially purified and cloned. P150 is part of the large subunit of the eukaryotic translation initiation factor 3 with sequence homology to centrosomin A. A significant correlation between p150 expression and malignancy in breast, cervical and esophageal cancer have recently been demonstrated. MATERIALS AND METHODS: Here, 110 colorectal carcinomas of different grades and stages, including lymph node and liver metastases were compared to adjacent normal mucosa by immunohistochemistry of P150. Western blot analysis of selected cases confirmed the expression levels determined by immunohistochemistry. Additionally, immuno-electron and laser scanning microscopy (LSM) was performed. RESULTS: All investigated carcinomas revealed high levels of p150 protein compared to normal adjacent mucosa. The staining intensity was slightly heterogeneous, and positivity was correlated to the tumor grade with statistically significant differences of p150 expression between normal and neoplastic mucosa (p<0.0001, Kruskal-Wallis test). Western blots confirmed higher expression levels of p150 in the tumor. Immunogold labelling and LSM investigation showed high expression levels of p150 on the rough endoplasmic reticulum and polyribosomes, indicating that p150 is translationally active in these tumors. CONCLUSION: Thus, we propose that p150 plays an important role in development and growth of colorectal carcinomas. Furthermore, p150 expression might provide us with reliable information on the biological behaviour of tumors and the clinical course of the disease.

Immunogold-labeled S-phase neoblasts, total neoblast number, their distribution, and evidence for arrested neoblasts in Macrostomum lignano (Platyhelminthes, Rhabditophora).

Neoblasts in Platyhelminthes are the only cells to proliferate and differentiate into all cell types. In Macrostomum lignano, the incorporation of 5'-bromo-2'-deoxyuridine (BrdU) in neoblasts confirmed the distribution of S-phase cells in two lateral bands. BrdU labeling for light and for transmission electron microscopy (TEM) identified three populations of proliferating cells: somatic neoblasts located between the epidermis and gastrodermis (mesodermal neoblasts), neoblasts located within the gastrodermis (gastrodermal neoblasts), and gonadal S-phase cells. In adults, three stages of mesodermal neoblasts (2, 2-3, and 3) defined by their ultrastructure were found. Stage 1 neoblasts where only seen in hatchlings. These stages either were phases within the S-phase of one neoblast pool or were subsequent stages of differentiating neoblasts, each with its own cell cycle. Regular TEM and immunogold labeling provided the basis for calculating the total number of neoblasts and the ratio of labeled to non-labeled neoblasts. Somatic neoblasts represented 6.5% of the total number of cells. Of these, 27% were labeled in S-phase. Of this fraction, 33% were in stage 2, 46% in stage 2-3, and 21% in stage 3. Immunogold labeling substantiated results concerning the differentiation of neoblasts into somatic cells. Non-labeled stage 2 neoblasts were present, even after a 2-week BrdU exposure. Double labeling of mitoses and FMRF-amide revealed a close spatial relationship of mesodermal neoblasts with the nervous system. Immunogold-labeled sections showed that nearly 70% of S-phase cells were in direct contact or within 5 microm from nerve cords.

Cell renewal and apoptosis in macrostomum sp. [Lignano].

In platyhelminths, all cell renewal is accomplished by totipotent stem cells (neoblasts). Tissue maintenance is achieved in a balance between cell proliferation and apoptosis. It is known that in Macrostomum sp. the epidermis undergoes extensive cell renewal. Here we show that parenchymal cells also exhibit a high rate of cell turnover. We demonstrate cell renewal using continuous 5'bromo-2-deoxyuridine (BrdU) exposure. About one-third of all cells are replaced after 14 days. The high level of replacement requires an equivalent removal of cells by apoptosis. Cell death is characterized using a combination of three methods: (1). terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), (2). specific binding of phosphatidyl-serine to fluorescent-labelled annexin V and (3). identification of apoptotic stages by ultrastructure. The number of cells observed in apoptosis is insufficient to explain the homeostasis of tissues in Macrostomum. Apoptosis-independent mechanisms may play an additional role in tissue dynamics.

Migration of dendritic cells into lymphatics-the Langerhans cell example: routes, regulation, and relevance.

Dendritic cells are leukocytes of bone marrow origin. They are central to the control of the immune response. Dendritic cells are highly specialized in processing and presenting antigens (microbes, proteins) to helper T lymphocytes. Thereby, they critically regulate further downstream processes such as the development of cytotoxic T lymphocytes, the production of antibodies by B lymphocytes, or the activation of macrophages. A new field of dendritic cell biology is the study of their potential role in inducing peripheral tolerance. The immunogenic/tolerogenic potential of dendritic cells is increasingly being utilized in immunotherapy, particularly for the elicitation of antitumor responses. One very important specialization of dendritic cells is their outstanding capacity to migrate from sites of antigen uptake to lymphoid organs. Much has been learned about this process from studying one particular type of dendritic cell, namely, the Langerhans cell of the epidermis. Therefore, the migratory properties of Langerhans cells are reviewed. Knowledge about this "prototype dendritic cell" may help researchers to understand migration of other types of dendritic cells.

Infection of the glass-eel swimbladder with the nematode Anguillicola crassus.

The ability of the nematode Anguillicola crassus to infect eel larvae (glass-eel stage) was tested. The results show that glass-eels fed on infected copepods, the natural intermediate host of the nematode, can be infected. Light microscopical examination of the infected developing swimbladder tissue revealed that the infection results in a significant thickening of the connective tissue. The basolateral labyrinth of gas gland cells is very much reduced in infected swimbladders, and the distance of gas gland cells to blood capillaries is enlarged. Critical swimming speed, defined as the speed where the larvae were no longer able to swim against the current, was similar in infected and uninfected animals. At intermediate speeds (about 60-80% of critical swimming speed) infected eels showed a slightly higher swimming activity than control animals. Resting oxygen consumption, measured as an index of metabolic activity, within the first 2 months of infection was higher in control animals, which may be due to a reduced rate of activity in infected glass-eels. By 4-5 months after the infection, however, it was significantly higher in infected animals. This may indicate that at this stage a higher activity of the animals is required to compensate for the increase in body density, but swimming performance of infected and non-infected glass-eels was not significantly different. Oxygen consumption during swimming activity, measured in a swim tunnel at 50% of maximal swimming speed, also was not affected. The results thus show that even glass-eels can be infected with A. crassus, and this probably contributes to the rapid spread of the nematode in Europe. While aerobic metabolism during swimming activity is not affected at this stage of infection, the swimbladder tissue shows severe histological changes, which most likely will impair swimbladder function.

Swim bladder gas gland cells produce surfactant: in vivo and in culture.

Electron microscopical examination of gas gland cells of the physostome European eel (Anguilla anguilla) and of the physoclist perch (Perca fluviatilis) revealed the presence of significant numbers of lamellar bodies, which are known to be involved in surfactant secretion. In the perch, in which the gas gland is a compact structure and gas gland cells are connected to the swim bladder lumen via small canals, lamellar bodies were also found in flattened cells forming the swim bladder epithelium. Flat epithelial cells are absent in the eel swim bladder, in which the whole epithelium consists of cuboidal gas gland cells. In both species, Western blot analysis using specific antibodies to human surfactant protein A (SP-A) showed a cross-reaction with swim bladder tissue homogenate proteins of approximately 65 kDa and in the eel occasionally of approximately 120 kDa, probably representing SP-A-like proteins in a dimeric and a tetrameric state. An additional band was observed at approximately 45 kDa. Western blots using antibodies to rat SP-D again resulted in a single band at approximately 45 kDa in both species, suggesting that there might be a cross-reaction of the antibody to human SP-A with an SP-D-like protein of the swim bladder tissue. To localize the surfactant protein, eel gas gland cells were cultured on permeable supports. Under these conditions, the gas gland cells regain their characteristic polarity. Electron microscopy confirmed the presence of lamellar bodies in cultured cells, and occasionally, exocytotic events were observed. Immunohistochemical staining using an antibody to human SP-A demonstrated the presence of surfactant protein only in luminal membranes and in adjacent lateral membranes. Only occasionally, evidence was found for the presence of surfactant protein in lamellar bodies.

Ultrastructural analysis of human endothelial cells after hypothermic storage in organ preservation solutions.

BACKGROUND: Protection of vascular endothelium is a critical factor in organ preservation for transplantation. This study aims at a morphological assessment of endothelial cell injury in a comparison of storage solutions, using a cell culture model of cold preservation and rewarming. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured in monolayer and exposed to hypothermic storage in University of Wisconsin (UW), histidine-tryptophane-ketoglutarate (HTK), and EuroCollins solutions for 6 h and subsequent rewarming for 30 min or 6 h. Alterations of subcellular structures and cell-cell contacts were analyzed by transmission electron microscopy (TEM) and light microscopic assessment after actin and nuclear staining. RESULTS: Structural alterations of mitochondria, endoplasmic reticulum, nuclei, and cytoskeletal fibers as well as disruption of intercellular contacts were found after cold storage in HTK and EuroCollins solutions. In contrast, storage in UW solution resulted in minimum changes of stress fibers only. A rapid rearrangement of structural alterations was achieved during rewarming in cell culture medium in all experimental groups. CONCLUSIONS: Preservation of endothelial cell structure is best achieved by UW solution. Ultrastructural cell damage is a direct consequence of hypothermic storage and is fully reversible during rewarming after short storage times.

Localization of carbonic anhydrase in swimbladder of European eel (Anguilla anguilla) and perch (Perca fluviatilis).

The distribution of carbonic anhydrase in swimbladder tissue and especially in gas gland cells of the European eel (Anguilla anguilla) and the perch (Perca fluviatilis) was analysed using histochemical staining according to Hansson (1967), with modifications proposed by Riddersträle (1991). While in the European eel, gas gland cells are distributed as a single layered epithelium over the whole secretory part of the swimbladder, the gas gland of the perch consists of a compact, richly vascularized 'multilayered' epithelium, in which gas gland cells have contact with the swimbladder lumen via small channels. In spite of these differences in organization, membranes of gas gland cells near blood vessels are richly folded in both species. A strong histochemical staining for carbonic anhydrase was observed in these membrane foldings. With prolonged incubation times a positive reaction was also observed in the cytoplasm of gas gland cells. In addition, the vascular endothelium and the erythrocytes showed a positive histochemical reaction. No staining, however, was visible in apical membranes towards the lumen of the swimbladder. In the perch, swimbladder epithelial cells outside the gas gland showed no positive staining of carbonic anhydrase. The results thus indicate that carbonic anhydrase activity is especially concentrated in membranes facing blood vessels. This suggests that a rapid equilibrium of the CO2/HCO3- reaction in the intracellular as well as in the extracellular space is essential for swimbladder function.

Development of the musculature in the limpet Patella (Mollusca, Patellogastropoda).

Whole-mount technique using fluorescent-labelled phalloidin for actin staining and confocal laser scanning microscopy as well as semi-thin serial sectioning, scanning and transmission electron microscopy were applied to investigate the ontogeny of the various muscular systems during larval development in the limpets Patella vulgata L. and P. caerulea L. In contrast to earlier studies, which described a single or two larval shell muscles, the pretorsional trochophore-like larva shows no less than four different muscle systems, namely the asymmetrical main head/foot larval retractor muscle, an accessory larval retractor with distinct insertion area, a circular prototroch/velar system, and a plexus-like pedal muscle system. In both Patella species only posttorsional larvae are able to retract into the shell and to close the aperture by means of the operculum. Shortly after torsion the two adult shell muscles originate independently in lateral positions, starting with two fine muscle fibres which insert at the operculum and laterally at the shell. During late larval development the main larval retractor and the accessory larval retractor become reduced and the velar muscle system is shed. In contrast, the paired adult shell muscles and the pedal muscle plexus increase in volume, and a new mantle musculature, the tentacular muscle system, and the buccal musculature arise. Because the adult shell muscles are entirely independent from the various larval muscular systems, several current hypotheses on the ontogeny and phylogeny of the early gastropod muscle system have to be reconsidered.

Purification and characterization of human sarcomeric mitochondrial creatine kinase.

In order to set the basis for detailed clinical investigations, mitochondrial creatine kinase (Mi-CK) was purified to homogeneity from human cardiac muscle. Biophysical characterization by SDS-PAGE, gel permeation chromatography and by electron microscopy of negatively stained single molecules demonstrated that, similar to other vertebrate Mi-CKs, human sarcomeric Mi-CK occurs in two different oligomeric forms, dimers and octamers, that are readily interconvertible. The apparent MTs of Mi-CK protomers, dimers and octamers were 43,600 +/- 800, 79,700 +/- 800 and 371,000 +/- 3000, respectively. In addition, isoelectric focussing proved to be a suitable technique for routinely distinguishing Mi-CK from cytosolic MM-CK and gave pl values of 8.30 +/- 0.04 and 7.44 +/- 0.04 for octameric and dimeric human sarcomeric Mi-CK. Circumstantial evidence suggests that both the highly symmetrical structure and the high pI value of Mi-CK octamers are crucial determinants for the physiological functions of this enzyme.

Fine structural and immunocytochemical studies on the eyeless aesthetes of Leptochiton algesirensis, with comparison to Leptochiton cancellatus (Mollusca, Polyplacophora).

The aesthetes of Leptochiton algesirensis (Capellini, 1859) and Leptochiton cancellatus (Sowerby, 1840) consist of six to eight microaesthetes surrounding one macroaesthete. The monocellular microaesthetes include many microtubules, neurosecretory vesicles, and unperforated, subsidiary caps. Basally they are in contact with tiny nerve processes via probably electrical synapses. Each macroaesthete consists of a perforated apical cap and various cell types: flattened peripheral cells, various types of mucous cells, and three or four monociliary sensory cells. Although lacking photoreceptors, the aesthetes of Leptochiton algesirensis combine storage-secretory and sensory functions. The latter function is confirmed by positive immunoreactions against (neuro-)tubulin and synaptophysine. The high degree of structural and functional similarity between polyplacophoran aesthetes and the analogous caeca of brachiopods is demonstrated.

Assessment of endothelial preservation in human cell cultures.

BACKGROUND: Impairment of microcirculation due to endothelial cell damage must be considered a limiting factor in organ preservation. The present study aims at a quantitative assessment of preservation-induced injury in cultured human endothelial cells. METHODS: Monolayer cultures of human umbilical vein endothelial cells were exposed to cold (40 degrees C) hypoxic storage in University of Wisconsin solution, histidine-tryptophane-ketoglutarate solution, Euro-Collins solution, and saline solution. Cellular integrity was evaluated by viable cell count, ultrastructural analysis, and prostacyclin release after 24, 48, and 72 hours of storage and subsequent 6 hours of reincubation in culture medium at 37 degrees C. Expression of intercellular adhesion molecule-1 was investigated after 6, 12, and 24 hours of cold preservation and after 6 hours of rewarming. RESULTS: Cellular viability was best maintained with University of Wisconsin and histidine-tryptophane-ketoglutarate solutions with no significant reduction of cell count up to 72 hours; Euro-Collins solution and saline solution caused a significant decline in cell numbers after 24 hours (p < 0.05). Morphology was best preserved by University of Wisconsin solution. Prostacyclin values were elevated after 24 hours in Euro-Collins solution and saline solution, after 48 hours in histidine-tryptophane-ketoglutarate, Euro-Collins, and saline solutions, and after 72 hours in Euro-Collins solution (p < 0.05, compared with University of Wisconsin solution). ICAM expression was weak after cold storage (24 hours) in University of Wisconsin solution, moderate after incubation in histidine-tryptophane-ketoglutarate and Euro-Collins solutions and intensive after storage in saline solution. In contrast, rewarming caused intensive expression of intercellular adhesion molecule-1 in all experimental groups as compared with controls, which showed baseline expression at any time. CONCLUSIONS: From our results we conclude that in this model cellular integrity is best protected by University of Wisconsin solution, increased prostacyclin release is consistent with morphologic alterations and intercellular adhesion molecule-1 expression is clearly up-regulated in endothelial cells under reperfusion conditions after cold hypoxic storage.

Differentiation of the body wall musculature in Macrostomum hystricinum marinum and Hoploplana inquilina (Plathelminthes), as models for muscle development in lower Spiralia.

Recent studies on the differentiation of the body wall musculature in a medicinal leech and in the free-living plathelminth Macrostomum hystricinum marinum, Beklemischev 1950 provide the first evidence of a complex developmental signalling pattern, possibly involving stem cells and the nervous system, in the organization of the muscle grid formed by developing myocytes. To enhance further our understanding of the ontogenetic and phylogenetic origin of such muscle grids, which consist of circular, longitudinal and diagonal muscle fibres, we have undertaken a study of muscle development in the polyclad flatworm Hoploplana inquilina Wheeler 1894 in collaboration with the Marine Biological Laboratory, Woods Hole. We have also continued our examination of the development of the body wall musculature in M. hystricinum. Both species were studied using rhodamine-phalloidin staining and transmission electron microscopy. Additional visualization of the fluorescent whole mount preparations was performed with confocal laser microscopy and digital image processing. The results of our investigation suggest that: (1) the mechanism of muscle development in H. inquilina supports the deeply rooted concept of bilateral symmetry (right and left longitudinal founder muscle), and (2) a first circular muscle in this species develops on the border between an anterior body unit and the main body; a caudalmost region is less obvious. The presence of a spiral muscle functioning as a circular muscle system of the "head region" points to a separate developmental mechanism for this region and the trunk. In contrast to H. inquilina, where the larval stage forces an intermediate restructuring of the musculature of the body wall before the adult body shape is finally developed, the formation of the body wall musculature of M. hystricinum already seems constrained by the adult body shape.

Ultrastructural analysis of thymic nurse cell epithelium.

Thymic nurse cells (TNC), a paradigmatic cell type of cortical epithelium, are large lymphoid-epithelial cell complexes of thymocytes enclosed within vacuoles lined by the epithelial cell membrane. TNC express major histocompatibility complex (MHC) class I and class II molecules on their surface and vacuole-lining membranes at high density and it was suggested that TNC provide an optimal microenvironment for positive selection of T cells. In this report we present electron microscopical data demonstrating that chicken TNC display morphological structures of exocytosis previously shown for hormone-secreting cells. In TNC, however, exocytosis is restricted to the capillary cleft between the epithelial cell and engulfed thymocytes. Thus, besides physical contact between the epithelial cell and enclosed thymocytes, TNC may additionally influence the development of thymocytes through release of soluble factors in a restricted microenvironment. By employing the 3-(2,4-dinitroanilino)-3'-amino-N-methyl-propylamine technique which at the ultrastructural level detects acidic organelles involved in processing of antigens presented by MHC class II molecules, we also show that TNC contain acidic compartments similar to classical antigen-presenting cells, i.e. early and late endosomes and lysosomes, albeit in a lower amount than in thymic dendritic cells. This fact provides evidence that TNC not only are capable of antigen presentation but also possess the intracellular machinery for antigen processing.