Light and electron microscopic studies on the influence of stress on prolactin-immunoreactivity in rat anterior pituitary lobe.

An increasing number of evidence suggests an important role of prolactin in the modulation of stress response. However, the mechanisms of its action on the HPA axis are not yet understood. Glucocorticoids, liberated from adrenal cortex due to hormonal signals from pituitary corticotrophs are known to play a key role in systemic stress response. Previously we found evidence that corticosteroid-binding globulin (CBG) is involved in rapid, membrane-mediated actions of adrenal steroids. Here we studied qualitatively immunostainings for prolactin and CBG in pituitaries of male rats that had been subjected to osmotic challenge. We also examined late pregnant, parturient and early lactating rats, assuming that parturition represents a strong physiological stress. We employed double immunofluorescencent staining of semithin sections and immunoelectron microscopy. In stressed males we found increased prolactin immunofluorescence associated with membranes while in controls this staining was predominantly cytoplasmatic. CBG immunofluorescence was found in almost all prolactin cells of stressed males while such double staining was only occasionally observed in controls. Similar observations were made in females: While parturient rats showed intense membrane associated double staining for both antigens, late pregnant and early lactating animals showed patterns similar to that of male controls. Immunoelectron microscopy revealed increased exocytosis of prolactin containing vesicles in lactating rats. CBG was localized on cell membranes and additionally within prolactin vesicles. Our observations suggest prolactin liberation from pituitary lactotrophs along with CBG upon systemic stress response. Membrane effects of glucocorticoids mediated by CBG may be linked to stimulus secretion of prolactin.

Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis.

Although cytotoxicity and endocytosis of nanoparticles have been the subject of numerous studies, investigations regarding exocytosis as an important mechanism to reduce intracellular nanoparticle accumulation are rather rare and there is a distinct lack of knowledge. The current study investigated the behavior of human microvascular endothelial cells to exocytose cerium dioxide (CeO2) nanoparticles (18.8 nm) by utilization of specific inhibitors [brefeldin A; nocodazole; methyl-ß-cyclodextrin (MßcD)] and different analytical methods (flow cytometry, transmission electron microscopy, inductively coupled plasma mass spectrometry). Overall, it was found that endothelial cells were able to release CeO2 nanoparticles via exocytosis after the migration of nanoparticle containing endosomes toward the plasma membrane. The exocytosis process occurred mainly by fusion of vesicular membranes with plasma membrane resulting in the discharge of vesicular content to extracellular environment. Nevertheless, it seems to be likely that nanoparticles present in the cytosol could leave the cells in a direct manner. MßcD treatment led to the strongest inhibition of the nanoparticle exocytosis indicating a significant role of the plasma membrane cholesterol content in the exocytosis process. Brefeldin A (inhibitor of Golgi-to-cell-surface-transport) caused a higher inhibitory effect on exocytosis than nocodazole (inhibitor of microtubules). Thus, the transfer from distal Golgi compartments to the cell surface influenced the exocytosis process of the CeO2 nanoparticles more than the microtubule-associated transport. In conclusion, endothelial cells, which came in contact with nanoparticles, e.g., after intravenously applied nano-based drugs, can regulate their intracellular nanoparticle amount, which is necessary to avoid adverse nanoparticle effects on cells.

Comparative evaluation of the impact on endothelial cells induced by different nanoparticle structures and functionalization.

In the research field of nanoparticles, many studies demonstrated a high impact of the shape, size and surface charge, which is determined by the functionalization, of nanoparticles on cell viability and internalization into cells. This work focused on the comparison of three different nanoparticle types to give a better insight into general rules determining the biocompatibility of gold, Janus and semiconductor (quantum dot) nanoparticles. Endothelial cells were subject of this study, since blood is the first barrier after intravenous nanoparticle application. In particular, stronger effects on the viability of endothelial cells were found for nanoparticles with an elongated shape in comparison to spherical ones. Furthermore, a positively charged nanoparticle surface (NH2, CyA) leads to the strongest reduction in cell viability, whereas neutral and negatively charged nanoparticles are highly biocompatible to endothelial cells. These findings are attributed to a rapid internalization of the NH2-functionalized nanoparticles in combination with the damage of intracellular membranes. Interestingly, the endocytotic pathway seems to be a size-dependent process whereas nanoparticles with a size of 20 nm are internalized by caveolae-mediated endocytosis and nanoparticles with a size of 40 nm are taken up by clathrin-mediated internalization and macropinocytosis. Our results can be summarized to formulate five general rules, which are further specified in the text and which determine the biocompatibility of nanoparticles on endothelial cells. Our findings will help to design new nanoparticles with optimized properties concerning biocompatibility and uptake behavior with respect to the respective intended application.

Anti-oxidative effects and harmlessness of asymmetric Au@Fe3O4 Janus particles on human blood cells.

The physical properties of asymmetric Janus particles are highly promising for future biomedical applications. However, only a few data is available on their biological impact on human cells. We investigated the biological impact of different Au@Fe3O4 Janus particle formulations in vitro to analyse specific uptake modalities and their potential cytotoxic effects on human cells of the blood regarding intravenous injection. We demonstrate that Au@Fe3O4 Janus particles exhibit a similar or even better biocompatibility compared to the well-studied spherical iron oxide nanoparticles. The impact of Janus particles on cells depends mainly on three factors. (1) Surface functionalization: NH2-functionalization of the Au or iron oxide domain induces a pronounced reduction of cell viability in contrast to non-functionalized variants which is caused by the damage of intracellular membranes. (2) The nature of the metal oxide component, greatly affects cell viability, as shown by a comparison with Au@MnO Janus particles. (3) The overall surface charge and the size of nanoparticles have a higher impact on internalization and cellular metabolism than the Janus character per se. Interestingly, Janus particle associated DNA damage is independent of the effects on the cellular ATP level. However, not only the structure and functionalization of the Janus particle surface determines the particle's adhesion and intracellular fate, but also the constitution of the cell surface as shown by different modification experiments. The multifactorial in vitro approach presented in this study demonstrated the high capability of the Janus particles. Especially Au@Fe3O4 Janus particles bear great potential for applications in vivo.

Ultrastructural and ultraimmunohistochemical changes upon partial nephrectomy and uranyl intoxication in the rat kidney.

We studied kidneys of rats intoxicated with uranylnitrate (UN) or subjected to 5/6 nephrectomy (NX) or after a combination of both procedures (NX-UN). Our observations indicate that UN causes impressive changes of ultrastructure (partial loss of brush border, appearance of intercellular clefts in the epithelial barrier) and altered protein expression (α-SMA, collagen I and III) in proximal tubule cells. Renal parameters (creatinine clearance, proteinuria) seemed to be unaffected. Blood pressure recovered to normal values within 12 months. However ultrastructural and functional restoration of modified proximal tubules was not complete. We conclude that changed proximal tubules may induce progression of interstitial fibrosis causing renal failure. NX animals and more pronounced NX-UN animals showed dramatic changes in renal function. We observed increased levels of proteinuria, blood pressure and decreased creatinine clearance. Progressive glomerular reorganization includes loss of filtration gaps and enhanced thickness of glomerular basement membranes (GBM) with increased immunoreactivity for collagen IV. Cells in vicinity of Bowman's capsule contained high amounts of immunoreactive α-smooth muscle actin. The NX-UN group showed more dramatic changes in ultrastructure of proximal tubules including apoptosis. Enhanced expression and secretion of extracellular matrix proteins (ECM e.g. collagens I, III, fibronectin) indicate progressive epithelial-mesenchymal transition (EMT) leading to permanent impairment of renal function.

Current knowledge on PB1-F2 of influenza A viruses.

Almost 10 years ago, an eleventh protein of influenza A viruses was discovered in a search for CD8+ T-cell epitopes. This protein was named PB1-F2 since it is encoded in the +1 reading frame of the PB1 gene segment. Various studies have shown that PB1-F2 has a pleiotropic effect: (1) The protein can induce apoptosis in a cell type-dependent manner, (2) PB1-F2 is able to promote inflammation, and (3) finally it up-regulates viral polymerase activity by its interaction with the PB1 subunit. These properties could contribute to an enhanced pathogenicity. However, the underlying mechanism is not fully understood yet. New data suggest that some effects of PB1-F2 are strain-specific and host-specific.

Characterization of iron oxide nanoparticles adsorbed with cisplatin for biomedical applications.

The aim of this study was to characterize the behaviour of cisplatin adsorbed magnetic nanoparticles (cis-MNPs) for minimal invasive cancer treatments in preliminary in vitro investigations. Cisplatin was adsorbed to magnetic nanoparticles (MNPs) by simple incubation. For stability determinations, cis-MNPs were incubated in dH(2)O, phosphate-buffered saline (PBS) and fetal calf serum (FCS) at 4-121 degrees C up to 20 weeks. Hydrodynamic diameters were measured using laser diffraction. The extent of cisplatin linkage was determined by atomic absorption spectrometry. The magnetite core size was assessed by vibrating sample magnetometry and transmission electron microscopy. The specific loss power (SLP) was measured in an alternating magnetic field. Our results showed that a maximum of 10.3 +/- 1.6 (dH(2)O), 10 +/- 1.6 (PBS) and 13.4 +/- 2.2 (FCS) mg cisplatin g(-1) Fe could be adsorbed to MNPs. With hyperthermal (42 degrees C) or thermal ablative (60 degrees C) temperatures, used for therapeutic approaches, cisplatin did not desorb from cis-MNPs in dH(2)O during incubation times of 180 or 30 min, respectively. In PBS and FCS, cisplatin amounts adsorbed to MNPs decreased rapidly to approximately 50% and 25% at these temperatures. This cisplatin release will be necessary for successful chemotherapeutic activity and should increase the therapeutic effect of magnetic heating treatment in medicinal applications. The hydrodynamic diameters of MNPs or cis-MNPs were around 70 nm and magnetization data showed superparamagnetic behaviour. The obtained mean core diameter was around 12 nm. The SLP of the sample was calculated to be 75.5 +/- 1.6 W g(-1). In conclusion, cis-MNPs exhibit advantageous features for a facilitated desorption of cisplatin in biological media and the heating potential is adequate for hyperthermic treatments. Therefore, even though further detailed investigations are still necessary, tentative use in local tumour therapies aiming at a specific chemotherapeutic release in combination with magnetic heating seems to be feasible in the long term.

Cytochemical demonstration of expression and distribution of non-glycosylated human lysosomal cathepsin S in HEK 293 cells.

The lysosomal cysteine protease cathepsin S is synthesized as inactive precursor at the rough endoplasmic reticulum (ER), further processed in the Golgi compartment and finally targeted to the lysosomes where it becomes activated by the proteolytic cleavage of the inhibitory propeptide. Biochemical studies with a non-glycosylated mutant of procathepsin S (plasma membrane binding at 2 degrees C, reuptake of secreted enzyme at 37 degrees C) led to the suggestion of an additional sorting motif in procathepsin S besides the classical Man-6-P recognition signal. In order to further confirm this suggestion on a morphological basis we performed a series of laser scanning confocal microscopy (CLSM) and electron microscopical analyses with HEK 293 cells expressing the mutant non-glycosylated procathepsin S. Immunolocalization with CLSM documented clearly a fine granular fluorescence in the paranuclear region of mutant expressing cells. Electron microscopy demonstrated the presence of cathepsin S immunoreactive deposits within cytosolic vacuoles (lysosomes), at the plasma membrane and in ER buds. These buds were also visible in the cytosol as well as in form of concentrated patches at the plasma membrane indicating the direct transport of (pro)cathepsin S from the ER to the cell surface.