Attenuation of NF-kappaB signaling response to UVB light during cellular senescence

The ability of cells to adapt to environmental stresses undergoes a progressive reduction during aging. NF-kappaB-mediated signaling is a major defensive system against various environmental challenges. The aim of this study was to find out whether replicative senescence affects the response of the NF-kappaB signaling pathway to UVB light in human WI-38 and IMR-90 fibroblasts. The exposure of early passage fibroblasts to UVB light inhibited the proliferation and induced a flat phenotype similar to that observed in replicatively senescent fibroblasts not exposed to UVB light. The UVB radiation dose used (153 mJ/cm2) did not induce apoptosis in either early or late passage WI-38 fibroblasts. UVB exposure induced a prominent activation of the NF-kappaB signaling pathway both in early and in late passage WI-38 and IMR-90 fibroblasts. Interestingly, the response to UVB light was significantly attenuated in late passage fibroblasts. This attenuation was most prominent in DNA binding activities of nuclear NF-kappaB complexes. Similar senescence-related attenuation was also observed in the DNA binding activities of nuclear AP-1 and Sp-1 factors after UVB treatment. Immunoblotting and -cytochemistry showed an increase in nuclear localization of p50 and p65 components of NF-kappaB complexes. Supershift experiments showed that the specific NF-kappaB complexes contain p50 and p65 protein components but not p52 and c-Rel proteins. Cytoplasmic IkappaBalpha showed a marked decrease at protein level but an increase in phosphorylation after UVB treatment. Transient transfection assays with TK5-CAT and TK10-CAT plasmids carrying NF-kappaB-responsive sites of the TNFalpha promoter were used to analyze the functional activity of the NF-kappaB complexes. Results showed that UVB exposure induced an increase in NF-kappaB-driven CAT expression both in early and in late passage fibroblasts though the response was significantly stronger in early passage fibroblasts. Our results show that the induction of NF-kappaB-mediated signaling by UVB light is highly attenuated in senescent fibroblasts. This attenuation may reduce the stress resistance during cellular senescence.     

Changes in leaf trichomes and epicuticular flavonoids during leaf development in three birch taxa

BACKGROUND AND AIMS: Changes in number of trichomes and in composition and concentrations of their exudates throughout leaf development may have important consequences for plant adaptation to abiotic and biotic factors. In the present study, seasonal changes in leaf trichomes and epicuticular flavonoid aglycones in three Finnish birch taxa (Betula pendula, B. pubescens ssp. pubescens, and B. pubescens ssp. czerepanovii) were followed. METHODS: Trichome number and ultrastructure were studied by means of light, scanning and transmission electron microscopy, while flavonoid aglycones in ethanolic leaf surface extracts were analysed by high-pressure liquid chromatography. KEY RESULTS: Density of both glandular and non-glandular trichomes decreased drastically with leaf expansion while the total number of trichomes per leaf remained constant, indicating that the final number of trichomes is established early in leaf development. Cells of glandular trichomes differentiate before those of the epidermis and produce secreted material only during the relatively short period (around 1-2 weeks) of leaf unfolding and expansion. In fully expanded leaves, glandular trichomes appeared to be at the post-secretory phase and function mainly as storage organs; they contained lipid droplets and osmiophilic material (probably phenolics). Concentrations (mg g(-1) d. wt) of surface flavonoids decreased with leaf age in all taxa. However, the changes in total amount ( microg per leaf) of flavonoids during leaf development were taxon-specific: no changes in B. pubescens ssp. czerepanovii, increase in B. pendula and in B. pubescens ssp. pubescens followed by the decline in the latter taxon. Concentrations of most of the individual leaf surface flavonoids correlated positively with the density of glandular trichomes within species, suggesting the participation of glandular trichomes in production of surface flavonoids. CONCLUSIONS: Rapid decline in the density of leaf trichomes and in the concentrations of flavonoid aglycones with leaf age suggests that the functional role of trichomes is likely to be most important at the early stages of birch leaf development.     

Microbiota Composition of the Intestinal Mucosa: Association with Fecal Microbiota?

The fecal and mucosal microbiota of infants with rectal bleeding and the fecal microbiota of healthy age-matched controls were investigated by fluorescent in situ hybridization. Bifidobacteria were the main genus in both the feces and mucosa. The other genera tested, Bacteroides, Clostridium, Escherichia coli and lactobacilli/enterococci, represented only minor constituents. No differences in fecal microbiota were observed between patients and controls. In the patients, however, four times greater numbers of bifidobacteria were observed in the feces when compared to the mucosa. Notwithstanding this difference, a strong positive correlation prevailed for bifidobacteria in feces and mucosal samples. The genera assessed accounted for 16% of total bacterial counts on mucosal samples and for 47% of total bacterial counts in feces. This indicates that the unidentified part of the microbiota, especially on the mucosa, deserves more attention.     

Comparative analysis of leaf trichome structure and composition of epicuticular flavonoids in Finnish birch species

The morphology, ultrastructure, density and distribution of trichomes on leaves of Betula pendula, B. pubescens ssp. pubescens, B. pubescens ssp. czerepanovii and B. nana were examined by means of light, scanning and transmission electron microscopy. The composition of flavonoids in ethanolic leaf surface extracts was analysed by high pressure liquid chromatography. All taxa examined contained both glandular and non-glandular trichomes (short and/or long hairs) but differed from each other in trichome ultrastructure, density and location on the leaf. Leaves of B. pubescens were more hairy than those of B. pendula, but the latter species had a higher density of glandular trichomes. Of the two subspecies of B. pubescens, leaves of ssp. pubescens had more short hairs on the leaf surface and four times the density of glandular trichomes of leaves of ssp. czerepanovii, whereas, in the latter subspecies, short hairs occurred largely on leaf veins, as in B. nana. The glandular trichomes were peltate glands, consisting of medullar and cortical cells, which differed structurally. Cortical cells possessed numerous small, poorly developed plastids and small vacuoles, whereas medullar cells had several large plastids with well-developed thylakoid systems and fewer vacuoles. In B. pubescens subspecies, vacuoles of the glandular cells contained osmiophilic deposits, which were probably phenolic, whereas in B. pendula, vacuoles of glandular trichomes were characterized by the presence of numerous myelin-like membranes. The composition of epicuticular flavonoids also differed among species. The two subspecies of B. pubescens and B. nana shared the same 12 compounds, but five of these occurred only in trace amounts in B. nana. Leaf surface extracts of B. pendula contained just six flavonoids, three of which occurred only in this species. In summary, the structure, density and distribution of leaf trichomes and the composition of epicuticular flavonoids represent good taxonomic markers for Finnish birch species.     

Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated

Twinfilin is a highly conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals. In addition to the previously characterized mammalian twinfilin-1, a second protein with approximately 65% sequence identity to twinfilin-1 exists in mouse and humans. However, previous studies failed to identify any actin binding activity in this protein (Rohwer, A., Kittstein, W., Marks, F., and Gschwendt, M. (1999) Eur. J. Biochem. 263, 518-525). Here we show that this protein, which we named twinfilin-2, is indeed an actin monomer-binding protein. Similar to twinfilin-1, mouse twinfilin-2 binds ADP-G-actin with a higher affinity (KD = 0.12 microM) than ATP-G-actin (KD = 1.96 microM) and efficiently inhibits actin filament assembly in vitro. Both mouse twinfilins inhibit the nucleotide exchange on actin monomers and directly interact with capping protein. Furthermore, the actin interactions of mouse twinfilin-1 and twinfilin-2 are inhibited by phosphatidylinositol (4,5)-bisphosphate. Although biochemically very similar, our Northern blots and in situ hybridizations show that these two proteins display distinct expression patterns. Twinfilin-1 is the major isoform in embryos and in most adult mouse non-muscle cell-types, whereas twinfilin-2 is the predominant isoform of adult heart and skeletal muscles. Studies with isoform-specific antibodies demonstrated that although the two proteins show similar localizations in unstimulated cells, they are regulated by different mechanisms. The small GTPases Rac1 and Cdc42 induce the redistribution of twinfilin-1 to membrane ruffles and cell-cell contacts, respectively, but do not affect the localization of twinfilin-2. Taken together, these data show that mammals have two twinfilin isoforms, which are differentially expressed and regulated through distinct cellular signaling pathways.     

Anaerobically digested poultry slaughterhouse wastes as fertiliser in agriculture

Chemical and physical analysis, 27-d plant growth assays with carrot (Daucus carota) and Chinese cabbage (Brassica campestris var. chinensis), and 5-d phytotoxicity assays with Chinese cabbage and perennial ryegrass (Lolium perenne) were used to investigate the suitability of anaerobically digested poultry slaughterhouse waste for fertiliser in agriculture and the effect of aerobic post-treatment on the properties of the digested material. The digested material appeared to be rich in nitrogen. In 27-d assays with digested material as nitrogen source, carrots grew almost as well as those fertilised with a commercial mineral fertiliser used as reference, whereas, the growth of Chinese cabbage was inhibited. In further 5-d phytotoxicity assays, the digested material inhibited the germination and root growth of ryegrass and Chinese cabbage, apparently because of organic acids present in it. Aerobic post-treatment of the material reduced its phytotoxicity but, probably due to the volatilisation of ammonia, resulted in loss of nitrogen.     

Stimulation of the secretion of pro-inflammatory cytokines by Bifidobacterium strains

To characterize the ability of bifidobacteria to affect the production of macrophage-derived cytokines, a murine macrophage-like cell line, J774.1, was cultured in the presence of 27 strains of heat-inactivated bifidobacteria. Bifidobacterium adolescentis and B. longum, known as adult-type bifidobacteria, induced significantly more pro-inflammatory cytokine secretion, IL-12 and TNF-alpha, by J774.1 cells, than did the infant-type bifidobacteria, B. bifidum, B. breve, and B. infantis (P<0.01). In contrast, B. adolescentis did not stimulate the production of anti-inflammatory IL-10 from J774.1 cells as the other tested bacteria did. The results suggest that the adult-type bifidobacteria, especially B. adolescentis, may be more potent to amplify but less able to down-regulate the inflammatory response.     

Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.     

Cleavage of the cyclin-dependent kinase 5 activator p35 to p25 does not induce tau hyperphosphorylation

Hyperphosphorylated tau protein is the primary component of neurofibrillary tangles observed in several neurodegenerative disorders. It has been hypothesized that in certain pathological conditions, the calcium activated protease, calpain, would cleave the cyclin-dependent kinase 5 (cdk5) activator p35 to a p25 fragment, which would lead to augmented cdk5 activity, and cdk5-mediated tau hyperphosphorylation. To test this hypothesis, we induced calpain-mediated p35 cleavage in rat hippocampal neuronal cultures and studied the relationship between p25 production, cdk5 activity, and tau phosphorylation. In glutamate-treated cells p35 was cleaved to p25 and this was associated with elevated cdk5 activity. However, tau phosphorylation was concomitantly decreased at multiple sites. The calpain inhibitor MDL28170 prevented the cleavage of p35 but had no effect on tau phosphorylation, suggesting that calpain-mediated processes, i.e., the cleavage of p35 to p25 and cdk5 activation, do not contribute to tau phosphorylation in these conditions. Treatment of the neuronal cultures with N-methyl-D-aspartic acid or with calcium ionophores resulted in an outcome highly similar to that of glutamate. We conclude that, in neuronal cells, the cleavage of p35 to p25 is associated with increased activity of cdk5 but not with tau hyperphosphorylation.     

Distributions of Li+, Na+, K+, Rb+, and Cs+ tracer ions in erythrocytes at 38 °C in relation to entry rates of these ions into cells at 0 °C

Forces that are able to transport Na+ and K+ into two compartments were investigated. A modified Nernst-Planck equation for coupled flows of electric current, water, and ions was integrated. The result shows that if alkali ions in the ion channel of the cell membrane are separated by their electric-current-induced inward flows against an electro-osmotic outward flow of water, the logarithms of the stationary cell/medium distributions of these ions should be proportional to the inverse of their diffusion mobilities. The relationship was tested in human erythrocytes. From inward and outward movements of tracer alkali ions, calculations were made to obtain their stationary distributions at infinite time. The cell/medium distributions determined in this way at 38 degrees C are Li+ = 0.59, 22Na+ = 0.044, 42K+ = 10.0, 86Rb+ = 11.9, and 137Cs+ = 3.07. The entry rates of ions into the cell at 0 degrees C are understood to represent their diffusion mobilities in the pump channel. The entry rates are Li+ = 1.44, 2Na+ = 1, 42K+ = 2.22, 86Rb+ = 2.39, and 137Cs+ = 1.72 relative to that of 22Na+. There is an expected negative correlation between the logarithms of the stationary cell/ medium distributions at 38 degrees C and the inverse of the entry rates into the cell at 0 degrees C for the five ions. It is suggested that the proposed physical forces cause the separation of alkali ions in the channel of Na,K-ATPase.