Morphological effects of ciliary neurotrophic factor treatment during neuromuscular synapse elimination

In adult skeletal muscles, exogenous ciliary neurotrophic factor (CNTF) induces axons and their nerve terminals to sprout. CNTF also regulates the amount of multiple innervation in developing skeletal muscles during synapse elimination, maintaining multiple innervation of muscle fibers. While CNTF may maintain multiple innervation by regulating developmental synapse elimination, it is also possible that CNTF induces the formation of new multiple innervation through a sprouting response. In this study I examined morphologically the effects of CNTF during synapse elimination in the extensor digitorum longus (EDL) muscle. Rat pups received injections of CNTF in one leg and vehicle in the other either early [postnatal day 7 (P7)-P13] or late (P14–P20) in development. The early treatment period corresponds to that time when the pattern of innervation in the EDL is converted from predominantly multiple to single innervation. The late treatment period is at the end of synapse elimination for the EDL but corresponds to the major period of synapse elimination in the levator ani (LA), allowing a comparison of effects on these two muscles from the same animals. On the day after the final injection, EDL muscles were dissected and stained with tetranitroblue tetrazolium and the resulting pattern of innervation was assessed. The present findings indicate that only the early CNTF treatment regulates the level of multiple innervation in the EDL. Moreover, the effect of early CNTF treatment was local, affecting multiple innervation only in the EDL from the CNTF-treated leg. CNTF injected during the late treatment period had no apparent effects on the EDL but had a potent effect on the pattern of innervation in the LA, significantly increasing the level of multiple innervation in this muscle. Thus, CNTF affected multiple innervation in these two muscles only if provided during the period when single innervation normally develops. There was no evidence to indicate that CNTF induced axons or their terminals to sprout during either treatment period. In conclusion, CNTF increases the level of multiple innervation, probably by regulating synapse elimination, and skeletal muscles themselves may be an important target site for CNTF action. Presumably, the sprouting response to CNTF found in adult muscle develops sometime after P21. © 1996 John Wiley & Sons, Inc.     

Massenentwicklungen von Algen und Wasserlinsen in der Talsperre Bleiloch (Thüringen) — gegenwärtige Situation,Ursachenanalyse und erste Ergebnisse zu deren Verringerung Algal Blooms and Duckweed Covers in the Bleiloch Reservoir (Thuringia) — Actually Situation,Causal Analysis and First Results of its Overcome

The effects of a decreased sewage release from a cellulose mill on the hypertrophic Bleiloch reservoir were studied. The waste water treatment reduced the concentration of yellow substances in the reservoir and resulted in a more light-coloured waterbody. It is estimated that the changes in the light attenuation in the Bleiloch reservoir after 1989 can be described as eutrophication. Selected physical, chemical, and biological parameters from 1994 show the effect of changes. Three large pelagic enclosures (diameter 10 m, length 20 m) were installed in the reservoir to assess effects of artifical mixing on phytoplankton biomass and composition. Two circulation techniques have been used in the enclosures (relase of compressed air at the bottom of the enclosure and mechanical mixing, where water was pumped from the surface downward). The investigation demonstrate that the magnitude of seasonal peaks of chlorophyll were depressed in the mixed enclosures during circulation. The results were discussed in comparison with a reference enclosure without mixing and the waterbody of the reservoir. Cryptophytes and other flagellates achieved dominance during mixing in the “air”-enclosure, while diatoms were the most abundant species in the „pump”︁-enclosure. The development of filamentous cyanobacteria, which are significant indicators of eutrophication, was disturbed in the enclosures. They were outcompeted by cryptophytes, movable chlorophytes and diatoms. Both mechanical pump and compressed air can be used to control excessive algal growth. Further studies on the dynamics of phytoplankton to artifical mixing are suggested. The results of further studies should design an in situ experiment in the reservoir to reduce algal growth by light limitation. Further on a dense duckweed cover spreads over the surface of the Bleiloch reservoir, which influences the light regime in the waterbody. Therefore factors influencing the duckweed growth were analysed. Special attention was focused on the influence of the pH, consequences of the mixing in the waterbody and the role of resting stages.     

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8⁺ cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both D^b class I MHC-restricted antigens and CD1d-restriced substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4^–CD8^– and CD8⁺ NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4^–CD8^–, but not CD8⁺ NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.Abbreviations CTLs cytotoxic T lymphocytes - NKT natural killer T - BM-DCs bone marrow-derived dendritic cells - CTM complete T-cell medium - FCS fetal calf serum - MMC mitomycin C - TCRs T cell receptors     

Metal-humic complexes and plant micronutrient uptake: a study based on different plant species cultivated in diverse soil types

There are several studies in the literature dealing with the effect of metal-humic complexes on plant metal uptake, but none of them correlate the physicochemical properties of the complexes with agronomic results. Our study covers both aspects under various experimental conditions. A humic extract (SHE) obtained from a sapric peat was selected for preparing the metal–humic complexes used in plant experiments. Fe–, Zn– and Cu–humic complexes with a reaction stoichiometry of 2:0.25 (humic:metal, w/w) were chosen after studying their stability and solubility with respect to pH (6–9) and the humic:metal reaction stoichiometry. Wheat and alfalfa plants were greenhouse cultured in pots containing one of three model soils: an acid, sandy soil and two alkaline, calcareous soils. Treatments were: control (no additions), SHE (53 mg kg^–1 of SHE), and metal (Cu, Zn and Fe)–SHE complexes (2.5 and 5 mg kg^–1 of metal rate and a SHE concentration to make 53 mg kg ^–1). Cu- and Zn–humic complexes significantly (p0.05) increased the plant uptake and the DTPA-extractable soil fraction of complexed micronutrients in most plant–soil systems. However, these effects were associated with significant increases (p0.05) of shoot and root dry weight only in alfalfa plants. In wheat, significant increases of root and shoot dry matter were only observed in the Cu–humic treated plants growing in the acid soil, where Cu deficiency was more intense. The Fe–humic complex did not increase Fe plant assimilation in any plant–soil system, but SHE increased Fe-uptake and/or DTPA-extractable soil Fe in the wheat–calcareous soil systems. These results, taken together with those obtained from the study of the pH- and SHE:metal ratio-dependent SHE complex solubility and stability, highlight the importance of the humic:Fe complex stoichiometry on iron bioavailability as a result of its influence on complex solubility.     

Synthesis and X-ray crystal structure of [Ag(phendio)<Subscript>2</Subscript>]ClO<Subscript>4</Subscript> (phendio = 1,10-phenanthroline-5,6-dione) and its effects on fungal and mammalian cells

The Cu(II) and Ag(I) complexes, [Cu(phendio)₃](ClO₄)₂4H₂O and [Ag(phendio)₂]ClO₄ (phendio = 1,10-phenanthroline-5,6-dione), are prepared in good yield by reacting phendio with the appropriate metal perchlorate salt. The X-ray crystal structure of the Ag(I) complex shows it to have a pseudo tetrahedral structure. `Metal-free' phendio and the Cu(II) and Ag(I) phendio complexes strongly inhibit the growth of the fungal pathogen Candida albicans, and are more active than their 1,10-phenanthroline analogues. The simple Ag(I) salts, AgCH<sub>3</sub>CO<sub>2</sub>, AgNO<sub>3</sub> and AgClO<sub>4</sub>.H<sub>2</sub>O display superior anti-fungal properties compared to analogous simple Cu(II) and Mn(II) salts, suggesting that the nature of the metal ion strongly influences activity. Exposing C. albicans to `metal-free' phendio, simple Ag(I) salts and [Ag(phendio)₂]ClO₄ causes extensive, non-specific DNA cleavage. `Metal-free' phendio and [Ag(phendio)₂]ClO₄ induce gross distortions in fungal cell morphology and there is evidence for disruption of cell division. Both drugs also exhibit high anti-cancer activity when tested against cultured mammalian cells.     

Patterns of divergence in the effects of mating on female reproductive performance in flour beetles

Sexual selection can lead to rapid divergence in reproductive characters. Recent studies have indicated that postmating events, such as sperm precedence, may play a key role in speciation. Here, we stress that other components of postmating sexual selection may be involved in the evolution of reproductive isolation. One of these is the reproductive investment made by females after mating (i.e., differential allocation). We performed an experiment designed to assess genetic divergence in the effects of mating on female reproductive performance in flour beetles, Tribolium castaneum. Females were mated to males of three different wild-type genotypes at two different frequencies, in all possible reciprocal combinations. Male genotype affected all aspects of female reproduction, through its effects on female longevity, total offspring production, reproductive rate, mating rate, and fertility. Moreover, male and female genotype interacted in their effects on offspring production and reproductive rate. We use the pattern of these interactions to discuss the evolutionary process of divergence and suggest that the pattern is most consistent with that expected if divergence was driven by sexually antagonistic coevolution. In particular, the fact that females exhibited a relatively weak response to males with which they were coevolved suggests that females have evolved resistance to male gonadotropic signals/stimuli.     

Characterization of the adhesive mucilages secreted by live diatom cells using atomic force microscopy

Atomic Force Microscopy (AFM) resolved the topography and mechanical properties of two distinct adhesive mucilages secreted by the marine, fouling diatom Craspedostauros australis. Tapping mode images of live cells revealed a soft and cohesive outer mucilage layer that encased most of the diatom's siliceous wall, and force curves revealed an adhesive force of 3.58 nN. High loading force, contact mode imaging resulted in cantilever 'cleaned' cell walls, which enabled the first direct observation of the active secretion of soft mucilage via pore openings. A second adhesive mucilage consisted of strands secreted at the raphe, a distinct slit in the silica wall involved in cell-substratum attachment and motility. Force measurements revealed a raphe adhesive strand(s) resistant to breaking forces up to 60 nN, and these strands could only be detached from the AFM cantilever probe using the manual stepper motor.     

Modelling the production and transport of dissolved organic carbon in forest soils

DyDOC describes soil carbon dynamics, with a focus on dissolved organic carbon (DOC). The model treats the soil as a three-horizon profile, and simulates metabolic carbon transformations, sorption reactions and water transport. Humic substances are partitioned into three fractions, one of which is immobile, while the other two (hydrophilic and hydrophobic) can pass into solution as DOC. DyDOC requires site-specific soil characteristics, and is driven by inputs of litter and water, and air and soil temperatures. The model operates on hourly and daily time steps, and can simulate carbon cycling over both long (hundreds-to-thousands of years) and short (daily) time scales. An important feature of DyDOC is the tracking of ¹⁴C, from its entry in litter to its loss as DO¹⁴C in drainage water, enabling information about C dynamics to be obtained from both long-term radioactive decay, and the characteristic ¹⁴C pulse caused by thermonuclear weapon testing during the 1960s ("bomb carbon"). Parameterisation is performed by assuming a current steady state. Values of a range of variables, including C pools, annual DOC fluxes, and ¹⁴C signals, are combined into objective functions for least-squares minimisation. DyDOC has been applied successfully to spruce forest sites at Birkenes (Norway) and Waldstein (Germany), and most of the parameters have similar values at the two sites. The results indicate that the supply of DOC from the surface soil horizon to percolating water depends upon the continual metabolic production of easily leached humic material. In contrast, concentrations and fluxes of DOC in the deeper soil horizons are controlled by sorption processes, involving comparatively large pools of leachable organic matter. Times to reach steady state are calculated to be several hundred years in the organic layer, and hundreds-to-thousands of years in the deeper mineral layers. It is estimated that DOC supplies 89% of the mineral soil carbon at Birkenes, and 73% at Waldstein. The model, parameterised with "steady state" data, simulates short-term variations in DOC concentrations and fluxes, and in DO¹⁴C, which are in approximate agreement with observations.     

Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice

To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2^−/− female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2^−/− mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2^−/−mammary fat pad showed a decrease in the content of myristic acid (C₁₄), a principal substrate for protein myristoylation and a potential peroxisomal β-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2^−/− mice.     

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.

Methods

The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.

Results

We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.

Conclusions

Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.

General significance

Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.