Kinetics of Langerhans cell chimerism in the skin of dogs following 2 Gy TBI allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Langerhans cells (LC) are bone marrow-derived cells in the skin. The LC donor/recipient chimerism is assumed to influence the incidence and severity of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). In nonmyeloablative (NM) HSCT the appearance of acute GVHD is delayed when compared with myeloablative conditioning. Therefore, we examined the development of LC chimerism in a NM canine HSCT model. METHODS: 2 Gy conditioned dogs received bone marrow from dog leukocyte antigen identical littermates. Skin biopsies were obtained pre- and post-transplant. LC isolation was performed by immunomagnetic separation and chimerism analysis by PCR analyzing variable-number-of-tandem-repeat markers with subsequent capillary electrophoresis. RESULTS: All dogs engrafted. Compared to peripheral blood chimerism the development of LC chimerism was delayed (earliest at day +56). None of the dogs achieved complete donor LC chimerism, although two dogs manifested a 100 % donor chimerism in peripheral blood at days +91 and +77. Of interest, one dog remained LC chimeric despite loss of donor chimerism in the peripheral blood cells. CONCLUSION: Our study indicates that LC donor chimerism correlates with chimerism development in the peripheral blood but occurs delayed following NM-HSCT.

Beneficial effect of the bioflavonoid quercetin on cholecystokinin-induced mitochondrial dysfunction in isolated rat pancreatic acinar cells.

The pathogenesis of acute pancreatitis (AP) is still poorly understood. Thus, a reliable pharmacological therapy is currently lacking. In recent years, an impairment of the energy metabolism of pancreatic acinar cells, caused by Ca(2+)-mediated depolarization of the inner mitochondrial membrane and a decreased ATP supply, has been implicated as an important pathological event. In this study, we investigated whether quercetin exerts protection against mitochondrial dysfunction. Following treatment with or without quercetin, rat pancreatic acinar cells were stimulated with supramaximal cholecystokinin-8 (CCK). CCK caused a decrease in the mitochondrial membrane potential (MMP) and ATP concentration, whereas the mitochondrial dehydrogenase activity was significantly increased. Quercetin treatment before CCK application exerted no protection on MMP but increased ATP to a normal level, leading to a continuous decrease in the dehydrogenase activity. The protective effect of quercetin on mitochondrial function was accompanied by a reduction in CCK-induced changes to the cell membrane. Concerning the molecular mechanism underlying the protective effect of quercetin, an increased AMP/ATP ratio suggests that the AMP-activated protein kinase system may be activated. In addition, quercetin strongly inhibited CCK-induced trypsin activity. The results indicate that the use of quercetin may be a therapeutic strategy for reducing the severity of AP.

Time-dependent decrement of dermal gadolinium deposits and significant improvement of skin symptoms in a patient with nephrogenic systemic fibrosis after temporary renal failure.

BACKGROUND: Nephrogenic systemic fibrosis (NSF) represents a rare fibrosing disorder occurring after administration of gadolinium-containing contrast agents during renal insufficiency. In order to prove the effect of gadolinium elimination on clinical signs, we identified and quantified gadolinium in skin biopsies of a 62-year-old patient with NSF with regard to improving skin lesions after recovery of renal function. METHODS: Gadolinium deposits were visualized and identified in NSF skin biopsies by light microscopy and transmission electron microscopy (EM) and by scanning EM. Inductively coupled plasma-mass spectrometry (ICPMS) was used for quantifying gadolinium concentration. RESULTS: Transmission EM studies revealed electron-dense material in connective matrix around blood vessels and inside lysosomes of histiocytes and fibroblasts. A remarkable reduction of gadolinium deposits was observed in transmission EM and scanning EM and confirmed by ICPMS in follow-up biopsies. After spontaneous recovery of renal function, his skin induration improved notably over the next 2 years. CONCLUSIONS: The reduction of clinical and histomorphological signs of NSF correlated with decreasing gadolinium concentration in skin biopsies within 3 years. Our study suggests a possible pathogenetic mechanism of NSF including a chance for recovery after elimination of gadolinium and reduced histamine liberation by mast cells.

Scanning electron microscopy of growing dental plaque: a quantitative study with different mouth rinses.

The aim of this study was to quantify the influence of different mouth rinses on dental plaque. Wearing splints with enamel pieces 24 volunteers rinsed with essential oils, amine/stannous fluoride, or chlorhexidine digluconate (0.12%) mouth rinses. After 24, 48, 72, and 96 h the enamel pieces were analyzed by scanning electron microscopy. The counts of cocci and bacilli in different plaque layers and the plaque thickness were almost similar using essential oils and amine/stannous fluoride. These results differed significantly from those of chlorhexidine digluconate mouth rinses. The results for plaque thickness were without significant differences between the groups at any appointment.

Detection of mercury in the 411-year-old beard hairs of the astronomer Tycho Brahe by elemental analysis in electron microscopy.

Hairs more than 400 years old of the famous astronomer Tycho Brahe were studied by electron microscopy to evaluate the hypothesis that Johannes Kepler murdered his teacher Brahe by mercury intoxication. The beard hairs showed a well-preserved ultrastructure with typical hair scales and melanosomes. The authors detected an accumulation of electron-dense granules of about 10 nm inside the outer hair scales, but not in the hair shaft and roots. At the places of these heavy-metal-containing granules they detected mercury besides other elements by energy dispersive X-ray analysis (EDX, Oxford, UK) in a field cathode scanning electron microscope (SEM, Gemini, Zeiss). The mercury-containing granules were found over the whole length of hairs, but only in the outer hair scales. Nevertheless, surface coatings of hairs were free of mercury. This distribution of mercury does not support the murder hypothesis, but could be related to precipitation of mercury dust from the air during long-term alchemistic activities.

Oxidative stress-induced cytotoxic and genotoxic effects of nano-sized titanium dioxide particles in human HaCaT keratinocytes.

Since nano-sized particles (NPs) are increasingly used in various fields of innovative biomedicine and industrial technologies, it is of importance to identify their potential human health risk. We investigated whether ROS-induced mitochondrial DNA damage is the mode of action of titanium dioxide-NPs (TiO2-NPs; ≤20 nm) to induce cytotoxic and genotoxic effects in human HaCaT keratinocytes in vitro. We showed that TiO2-NPs accumulate at the cell surface and are taken up by endocytosis. Micronucleus (MN) formation was found to be significantly maximal increased 24 h after treatment with 10 µg/ml and 48 h after treatment with 5 µg/ml TiO2-NPs about 1.8-fold respectively 2.2-fold of control. Mitochondrial DNA damage measured as "common deletion" was observed to be significantly 14-fold increased 72 h after treatment with 10 µg/ml TiO2-NPs when compared to control. Four hours after treatment with 5 and 50 µg/ml TiO2-NPs the level of ROS in HaCaT cells was found to be significantly increased about 7.5-fold respectively 16.7-fold of control. In conclusion, for the first time we demonstrate the induction of the mitochondrial "common deletion" in HaCaT cells following exposure to TiO2-NPs, which strongly suggests a ROS-mediated cytotoxic and genotoxic potential of NPs. However, the effects of the modification of TiO2-NPs, such as agglomeration, size distribution pattern and exposure time have to be further critically examined.

Ceramic honeycomb as support for covalent immobilization of laccase from Trametes versicolor and transformation of nuclear fast red.

The covalent immobilization of laccase on an inorganic ceramic support was investigated. The intention was to find a system of enzyme and reactor for a universal immobilization procedure. Laccase from Trametes versicolor as model enzyme was chosen. The special honeycomb structure of the monolith can be applied for intensive mixing of the reaction compounds. An appropriate reactor with ceramic material was constructed allowing different setup for enzyme immobilization and its application. To test the success of the immobilization, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) was used. The immobilized laccase was found to be stable over a time period of over 3 months. As an example for possible application for treatment of wastewater containing dyes, the conversion of nuclear fast red as model substrate was tested.

Multi-walled carbon nanotubes induce oxidative stress and apoptosis in human lung cancer cell line-A549.

Multi-walled carbon-nanotubes (MWCNTs)-induced apoptotic changes were studied in human lung epithelium cell line-A549. Non-cytotoxic doses of MWCNTs were identified using tetrazolium bromide salt (MTT) and lactate dehydrogenase (LDH) release assays. Cells were exposed to MWCNTs (0.5-100 µg/ml) for 6-72 h. Internalization and characterization of CNTs was performed by electron microscopy. Apoptotic changes were estimated by nuclear condensation, DNA laddering, and confirmed by expression of associated markers: p(53), p(21WAF1/CIP1), Bax, Bcl(2) and activated caspase-3. MWCNTs induced the production of reactive oxygen species and malondialdehyde along with significant decrease in the activity of catalase and glutathione. MWCNTs-induced ROS generation was found not to be associated with the mitochondrial activity. In general, the changes were significant at 10 and 50 µg/ml only. Results indicate the involvement of oxidative stress and apoptosis in A549 cells exposed to MWCNTs. Our studies provide insights of the mechanisms involved in MWCNTs-induced apoptosis at cellular level.

Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells.

BACKGROUND: 3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenviroment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them. METHODS: In this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay. RESULTS: Atomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures. CONCLUSIONS: Taken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3D-scaffolds protocols prior to in vivo engraftment of stem and progenitor cells in the context of regenerative medicine.

Nanoparticles induce changes of the electrical activity of neuronal networks on microelectrode array neurochips.

BACKGROUND: Nanomaterials are extensively used in industry and daily life, but little is known about possible health effects. An intensified research regarding toxicity of nanomaterials is urgently needed. Several studies have demonstrated that nanoparticles (NPs; diameter < 100 nm) can be transported to the central nervous system; however, interference of NPs with the electrical activity of neurons has not yet been shown. OBJECTIVES/METHODS: We investigated the acute electrophysiological effects of carbon black (CB), hematite (Fe2O3), and titanium dioxide (TiO2) NPs in primary murine cortical networks on microelectrode array (MEA) neurochips. Uptake of NPs was studied by transmission electron microscopy (TEM), and intracellular formation of reactive oxygen species (ROS) was studied by flow cytometry. RESULTS: The multiparametric assessment of electrical activity changes caused by the NPs revealed an NP-specific and concentration-dependent inhibition of the firing patterns. The number of action potentials and the frequency of their patterns (spike and burst rates) showed a significant particle-dependent decrease and significant differences in potency. Further, we detected the uptake of CB, Fe2O3, and TiO2 into glial cells and neurons by TEM. Additionally, 24 hr exposure to TiO2 NPs caused intracellular formation of ROS in neuronal and glial cells, whereas exposure to CB and Fe2O3 NPs up to a concentration of 10 µg/cm2 did not induce significant changes in free radical levels. CONCLUSION: NPs at low particle concentrations are able to exhibit a neurotoxic effect by disturbing the electrical activity of neuronal networks, but the underlying mechanisms depend on the particle type.

The mtDNA nt7778 G/T polymorphism affects autoimmune diseases and reproductive performance in the mouse.

Mitochondria are organelles of all nucleated cells, and variations in mtDNA sequence affect a wide spectrum of human diseases. However, animal models for mtDNA-associated diseases are rare, making it challenging to explore mechanisms underlying the contribution of mitochondria. Here, we identify a polymorphism in the mitochondrial genome, G-to-T at position 7778, which results in an aspartic acid-to-tyrosine (D-Y) substitution in the fifth amino acid of the highly conserved N-terminus of ATP synthase 8 (ATP8). Using a series of conplastic strains we show that this polymorphism increases susceptibility to multiple autoimmune diseases, including collagen-induced arthritis, autoimmune diabetes, nephritis and autoimmune pancreatitis. In addition, it impairs reproductive performance in females, but only in the MRL/MpJ strain. We also demonstrate that the mtAtp8 polymorphism alters mitochondrial performance, increasing H(2)O(2) production and affecting mitochondrial structure. Functional analysis reveals that the polymorphism increase the CD4 T cell adaptive potential to an oxidative phosphorylation impaired condition. Our findings provide direct experimental evidence for the role of mitochondria in autoimmunity and reproduction.

Calpain-mediated breakdown of cytoskeletal proteins contributes to cholecystokinin-induced damage of rat pancreatic acini.

The cytosolic cysteine protease calpain is implicated in a multitude of cellular functions but also plays a role in cell damage. Our previous results suggest that an activation of calpain accompanied by a decrease in its endogenous inhibitor calpastatin may contribute to pancreatic damage during cerulein-induced acute pancreatitis. The present study aimed at the time course of secretagogue-induced calpain activation and cellular substrates of the protease. Isolated rat pancreatic acini were incubated with a supramaximal concentration of cholecystokinin (0.1 microM CCK) for 30 min in the presence or absence of the calpain inhibitor Z-Val-Phe methyl ester (100 microM ZVP). The activation of calpain and the expression of calpastatin and the actin cytoskeleton-associated proteins alphaII-spectrin, E-cadherin and vinculin were studied by immunoblotting. The cell damage was assessed by lactate dehydrogenase release and ultrastructural analysis including fluorescence-labelled actin filaments. Immediately after administration, CCK led to activation of both calpain isoforms, mu- and m-calpain. The protease activation was accompanied by a decrease in the E-cadherin level and formation of calpain-specific breakdown products of alphaII-spectrin. A calpain-specific cleavage product of vinculin appeared concomitantly with changes in the actin filament organization. No effect of CCK on calpastatin was found. Inhibition of calpain by ZVP reduced CCK-induced damage of the actin-associated proteins and the cellular ultrastructure including the actin cytoskeleton. The results suggest that CCK-induced acinar cell damage requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease.

Galectin-1 induced activation of the mitochondrial apoptotic pathway: evidence for a connection between death-receptor and mitochondrial pathways in human Jurkat T lymphocytes.

Galectin-1 (gal-1) triggers T cell death by several distinct intracellular pathways including the activation of the death-receptor pathway. The aim of this study was to investigate whether gal-1 induced activation of the death-receptor pathway in Jurkat T lymphocytes mediates apoptosis via the mitochondrial pathway linked by truncated Bid (tBid). We demonstrate that gal-1 induced proteolytic cleavage of the death agonist Bid, a member of the Bcl-2/Bcl-xL family and a substrate of activated caspase-8, was inhibited by caspase-8 inhibitor II (Z-IETD-FMK). Downstream of Bid, gal-1 stimulated mitochondrial cytochrome c release as well as the activation and proteolytic processing of initiator procaspase-9 were effectively decreased by caspase-8 inhibitor II. Blocking of gal-1 induced cleavage of effector procaspase-3 by caspase-8 inhibitor II as well as by caspase-9 inhibitors I (Z-LEHD-FMK) and III (Ac-LEHD-CMK) indicates that receptor and mitochondrial pathways converged in procaspase-3 activation and contribute to proteolytic processing of effector procaspase-6 and -7. Western blot analyses and immunofluorescence staining revealed that exposure of Jurkat T cells to gal-1 resulted in the cleavage of the DNA-repair enzyme poly (ADP-ribose) polymerase, cytoskeletal alpha-fodrin, and nuclear lamin A as substrates of activated caspases. Our data demonstrate that Bid provides a connection between the death receptor and the mitochondrial pathway of gal-1 induced apoptosis in human Jurkat T lymphocytes.

Inhibitory effect of the lectin wheat germ agglutinin (WGA) on the proliferation of AR42J cells.

The rat pancreatic acinar tumour cell line AR42J is a widely used model to study the secretion, proliferation and differentiation of cells under the influence of hormones. These so-called amphicrine cells synthesize and secrete digestive enzymes as well as neuroendocrine peptides. They possess both subtypes of the highly glycosylated cholecystokinin (CCK) receptor which are important for the regulation of secretion and for cell growth. AR42J cells extrude CCK and gastrin-like hormone peptides and have the ability of an autostimulation (autocrine loop). The lectins wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I) bind to the glycosylated sites of these CCK receptors with the effect inhibiting CCK binding and thus inhibiting the CCK-induced Ca2+ release and alpha-amylase secretion. The so-called trophic hormones CCK and gastrin stimulate the secretion and proliferation of AR42J cells within the autocrine loop via autostimulation of their CCK receptors. In preceding papers, we described the inhibitory effect of WGA on the binding of 125I-CCK-8s to the CCK-A and -B receptors and the subsequent enzyme secretion of AR42J cells. In the present work, we studied the influence of the lectins WGA, UEA-I and galectin-1, as well as of the lectin-like enzyme alpha-amylase, on the proliferation of AR42J cells and prevention of autostimulation. The proliferation inhibition of the growth fraction was measured by estimation of the S-phase fraction by DNA flow cytometry. Whereas WGA inhibited the growth fraction significantly, UEA-I, human galectin-1 and human alpha-amylase had no significant effect. In transmission electron microscopy, we observed the accumulation of typical zymogen granules under the effect of WGA and a better differentiation of cells.

Mode of expression and functional characterization of FCT-3 pilus region-encoded proteins in Streptococcus pyogenes serotype M49.

The human pathogen Streptococcus pyogenes (group A streptococcus [GAS]) pilus components, suggested to play a role in pathogenesis, are encoded in the variable FCT (fibronectin- and collagen-binding T-antigen) region. We investigated the functions of sortase A (SrtA), sortase C2 (SrtC2), and the FctA protein of the most prevalent type 3 FCT region from a serotype M49 strain. Although it is considered a housekeeping sortase, SrtA's activity is involved in pilus formation in addition to its essentiality for GAS extracellular matrix protein binding, host cell adherence/internalization, survival in human blood, and biofilm formation. SrtC2 activity is crucial for pilus formation but dispensable for the other phenotypes tested in vitro. FctA is the major pilus backbone protein, simultaneously acting as the M49 T antigen, and requires SrtC2 and LepA, a signal peptidase I homologue, for monomeric surface expression and polymerization, respectively. Collagen-binding protein Cpa expression supports pilus formation at the pilus base. Immunofluorescence microscopy and fluorescence-activated cell sorting analysis revealed several unexpected expression patterns, as follows: (i) the monomeric pilus protein FctA was found exclusively at the old poles of GAS cells, (ii) FctA protein expression increased with lower temperatures, and (iii) FctA protein expression was restricted to 20 to 50% of a given GAS M49 population, suggesting regulation by a bistability mode. Notably, disruption of pilus assembly by sortase deletion rendered GAS serotype M49 significantly more aggressive in a dermonecrotic mouse infection model, indicating that sortase activity and, consequently, pilus expression allow a subpopulation of this GAS serotype to be less aggressive. Thus, pilus expression may not be a virulence attribute of GAS per se.

Prevention of lens epithelial cell growth in vitro using mibefradil-containing PLGA micro particles.

The prevention of the posterior capsule opacification is still unsolved. To interfere with proliferating cells the T-type calcium channel antagonist Mibefradil was immobilized in poly-lactic-co-glycolic-acid micro particles which were fixed at a capsular tension ring and tested in a human organ culture model as well as in human lens cells HLE-B3 in vitro. It is feasible to get a release significantly affecting cell viability and growth evaluated by MTT test and cell cycle analysis. In addition, Bionas(®) sensor chips were used for time-dependent adhesion experiments in living lens cells. Interestingly, the concentration of Mibefradil which inhibited subconfluent cells is not effective in confluent cells. This is an important feature for the protection of the intact tissue in the eye.

Detection of silver sulfide deposits in the skin of patients with argyria after long-term use of silver-containing drugs.

Five patients with generalized slate-gray discoloration of the skin have been diagnosed histologically as argyria in the last 35 years in the Department of Dermatology and Venereology of Rostock and Halle. Light microscopically, there was visible black pigmentation in histiocytes, fibroblasts, and multinucleated giant cells of the dermis. In the transmission electron microscope (TEM), the authors observed electron-dense deposits inside lysosomes and residual bodies of phagocytes as well as outside the cells in the connective matrix. These deposits were identified by elemental analysis in TEM and electron energy loss spectroscopy (EELS) as well as scanning electron microscope (SEM) and energy dispersive x-ray analysis (EDX) containing silver and sulfur. Therefore, they seem to consist of silver sulfide. Argyria is of low medical relevance and is very rarely induced because of silver-containing drugs. Nevertheless, there are still a lot of silver products on the market, easily available over-the-counter. Therefore, argyria should not be forgotten or missed in the diagnostics of human dermis.

Ischemic preconditioning prevents skeletal muscle tissue injury, but not nerve lesion upon tourniquet-induced ischemia.

BACKGROUND: Prolonged ischemia followed by reperfusion (I/R) of skeletal muscle results in significant tissue injury. Ischemic preconditioning (IPC), achieved by brief periods of ischemia before sustained ischemia, has been shown to ameliorate I/R injury in a variety of tissues. We demonstrate that tourniquet hind limb ischemia-induced injury of the muscle benefits from IPC, whereas the peripheral nerve suffers from prolonged ischemia time and mechanical deterioration on IPC. METHODS: In anesthetized rats, hind limb ischemia was induced by tourniquet for 3 hours followed by 24 hours of reperfusion. In an additional series of experiments, IPC (three cycles of 10 minutes I/10 minutes R) preceded hind limb ischemia. Sham-operated animals without ischemia served as controls. Skeletal muscle tissue injury was assessed with respect to microcirculation, inflammatory cell response, and cell integrity using intravital fluorescence microscopy, Western blot protein analysis, and tissue histochemistry. Analysis of tactile and thermal allodynia served as indicators for postischemic pain. In addition, motor nerve conduction velocity and transmission electron microscopy allowed assessing postischemic nerve lesion. RESULTS: Tourniquet of the hind limb caused marked perfusion failure, enhanced leukocyte-endothelial cell interaction, and apoptotic cell death. IPC was able to improve microvascular perfusion and to reduce inflammatory cell response. Of interest, apoptotic cell death, assessed by cell nuclear morphology in vivo as well as Western blot and immunohistochemical analysis of caspase-3 cleavage, can be substantially reduced by IPC in tourniquet ischemia of the hind limb. Application of the tourniquet abolished nerve conduction in all animals. Non-IPC-treated animals still showed tactile allodynia, whereas IPC further caused loss of pain sensation and motor function of the postischemic hind limb. CONCLUSIONS: High susceptibility of the peripheral nerve to compression-induced ischemic injury disproves IPC in its clinical application for surgical procedures requiring prolonged tourniquet ischemia.

Long-term response toward inorganic carbon limitation in wild type and glycolate turnover mutants of the cyanobacterium Synechocystis sp. strain PCC 6803.

Concerted changes in the transcriptional pattern and physiological traits that result from long-term (here defined as up to 24 h) limitation of inorganic carbon (C(i)) have been investigated for the cyanobacterium Synechocystis sp. strain PCC 6803. Results from reverse transcription-polymerase chain reaction and genome-wide DNA microarray analyses indicated stable up-regulation of genes for inducible CO(2) and HCO(3)(-) uptake systems and of the rfb cluster that encodes enzymes involved in outer cell wall polysaccharide synthesis. Coordinated up-regulation of photosystem I genes was further found and supported by a higher photosystem I content and activity under low C(i) (LC) conditions. Bacterial-type glycerate pathway genes were induced by LC conditions, in contrast to the genes for the plant-like photorespiratory C2 cycle. Down-regulation was observed for nitrate assimilation genes and surprisingly also for almost all carboxysomal proteins. However, for the latter the observed elongation of the half-life time of the large subunit of Rubisco protein may render compensation. Mutants defective in glycolate turnover (DeltaglcD and DeltagcvT) showed some transcriptional changes under high C(i) conditions that are characteristic for LC conditions in wild-type cells, like a modest down-regulation of carboxysomal genes. Properties under LC conditions were comparable to LC wild type, including the strong response of genes encoding inducible high-affinity C(i) uptake systems. Electron microscopy revealed a conspicuous increase in number of carboxysomes per cell in mutant DeltaglcD already under high C(i) conditions. These data indicate that an increased level of photorespiratory intermediates may affect carboxysomal components but does not intervene with the expression of majority of LC inducible genes.

Actin is not required for nanotubular protrusions of primary astrocytes grown on metal nano-lawn.

We used sub-micron metal rod decorated surfaces, 'nano-lawn' structures, as a substrate to study cell-to-cell and cell-to-surface interactions of primary murine astrocytes. These cells form thin membranous tubes with diameters of less than 100 nm and a length of several microns, which make contact to neighboring cells and the substrate during differentiation. While membrane protrusions grow on top of the nano-lawn pillars, nuclei sink to the bottom of the substrate. We observed gondola-like structures along those tubes, suggestive of their function as transport vehicles. Elements of the cytoskeleton such as actin fibers are commonly believed to be essential for triggering the onset and growth of tubular membrane protrusions. A rope-pulling mechanism along actin fibers has recently been proposed to account for the transport or exchange of cellular material between cells. We present evidence for a complementary mechanism that promotes growth and stabilization of the observed tubular protrusions of cell membranes. This mechanism does not require active involvement of actin fibers as the formation of membrane protrusions could not be prevented by suppressing polymerization of actin by latrunculin B. Also theoretically, actin fibers are not essential for the growing and stability of nanotubes since curvature-driven self-assembly of interacting anisotropic raft elements is sufficient for the spontaneous formation of thin nano-tubular membrane protrusions.