Spontaneously immortalized mouse embryo fibroblasts: growth behavior of wild-type and vimentin-deficient cells in relation to mitochondrial structure and activity

Dependent on the presence or absence of vimentin, primary mouse embryo fibroblasts exhibit different growth characteristics in vitro. While most Vim(+/+) fibroblasts stop dividing and die via apoptosis, a substantial fraction of cells immortalize and proliferate almost normally. Vim(-/-) fibroblasts cease to divide earlier, immortalize in vanishingly small numbers and thereafter proliferate extremely slowly. Early after immortalization, Vim(+/+) (imm) fibroblasts appear structurally almost normal, whereas Vim(-/-) (imm) fibroblasts equal postmitotic "crisis" cells, which are characterized by increased cell size, altered cell ultrastructure, nuclear enlargement, genome destabilization, structural degeneration of mitochondria, and diminution of mitochondrial respiratory activity. The differences between immortalized Vim(+/+) (imm) and Vim(-/-) (imm) fibroblasts persist during early cell cloning but disappear during serial subcultivation. At high cell passage, cloned, immortalized vim(-) fibroblasts grow nearly as fast as their cloned vim(+) counterparts, and also resemble them in size, ultrastructure, nuclear volume, and mitochondrial complement; they very likely employ redundancy to cope with the loss of vimentin function when adjusting structure and behavior to that of immortalized vim(+) fibroblasts. Reduction in nuclear size occurs via release of large amounts of filamentous chromatin into extracellular space; because it is complexed with extracellular matrix proteins, it tends to form clusters and to tightly stick to the surface of other cells, thus providing a potential for horizontal gene transfer. On the other hand, cloned vim(+) and vim(-) fibroblasts are equal in showing contact inhibition at young age and becoming anchorage-independent during serial subcultivation, as indicated by the formation of multilayered and -faceted cell sheets and huge spheroids on top of or in soft agar. With this, immortalized vim(-) fibroblasts reduce their adhesiveness to the substratum which, in their precrisis state and early after cloning, is much higher than that of their vim(+) counterparts. In addition, the coupling between the mitochondrial respiratory chain and oxidative phosphorylation is stronger in vim(+) than vim(-) fibroblasts. It appears from these data that after explantation of fibroblasts from the mouse embryo the primary cause of cell and mitochondrial degeneration, including genomic instability, is the mitochondrial production of reactive oxygen species in a vicious circle, and that vimentin provides partial protection from oxidative damage. As a matrix protein with specific in vitro and in vivo affinities for nuclear and mitochondrial, recombinogenic DNA, it may exert this effect preferentially at the genome level via its influence on recombination and repair processes, and in this way also assist the cells in immortalizing. Additional protection of mitochondria by vimentin may occur at the level of mitochondrial fatty acid metabolism.     

Soft shoreline engineering survey of ecological effectiveness

Historically, many urban waterfront shorelines were stabilized using hard shoreline engineering to protect developments from flooding and erosion, or to accommodate commercial navigation or industry. Today, there is growing interest in developing shorelines using ecological principles and practices that enhance habitat and improve aesthetics, while at the same time reducing erosion, providing stability, and ensuring shoreline safety (i.e., soft shoreline engineering). In 2008-2009, a survey of 38 soft shoreline engineering projects in the Detroit River-western Lake Erie watershed was conducted. In total, $17.3 million (combined U.S. and Canadian currency) was spent on these projects. Of the 38 projects implemented, six (16%) had some quantitative assessment of ecological effectiveness, while the remaining 32 lacked monitoring or only had qualitative assessment through visual inspection. Key lessons learned include: involve habitat experts at the initial stages of waterfront planning; establish broad-based goals with quantitative targets to measure project success; ensure multidisciplinary project support; start with demonstration projects and attract partners; treat habitat modification projects as experiments that promote learning; involve citizen scientists, volunteers, and universities in monitoring, and obtain post-project monitoring commitments up front in project planning; measure benefits and communicate successes; and promote education and outreach, including public events that showcase results and communicate benefits.     

Comparative analysis of the zeta-crystallin/quinone reductase gene in guinea pig and mouse

zeta-Crystallin is a novel nicotinamide adenine dinucleotide phosphate:quinone reductase, present at enzymatic levels in various tissues of different species, which is highly expressed in the lens of some hystricomorph rodents and camelids. We report here the complementary DNA (cDNA) cloning of zeta-crystallin from liver libraries in guinea pig (Cavia porcellus), where zeta-crystallin is highly expressed in the lens, and in the laboratory mouse (Mus musculus), where expression in the lens occurs only at enzymatic levels. A 5' untranslated sequence different from the one previously reported for the guinea pig lens cDNA was found in these clones. We also report the isolation of genomic clones including the complete guinea pig zeta-crystallin gene and the 5' region of this gene in mouse. These results show the presence of two promoters in the guinea pig zeta-crystallin gene, one responsible for expression at enzymatic levels and the other responsible for the high expression in the lens. The guinea pig lens promoter is not present in the mouse gene. This is the first example in which the recruitment of an enzyme as a lens crystallin can be explained by the acquisition of an alternative lens- specific promoter.      

Sequence of morphological and physiological events during natural ageing and senescence of a castor bean leaf: sieve tube occlusion and carbohydrate back-up precede chlorophyll degradation

The development of castor bean ( Ricinus communis L. var. sanguineus) leaves from bud break to abscission was studied to determine whether senescence of phloem precedes or follows chlorophyll degradation in the course of natural ageing of leaves. The castor bean leaf blade took 20 days for full expansion and its average life span was 60 days. From the day of full expansion on it suffered a substantial loss in N, a small loss in C, K and P and a gain in Ca, Mg and S. The content of soluble sugars increased with time, paralleled by a decrease of photosynthetic activity. Starch accumulated shortly before chlorophyll breakdown. The amino acid level in the leaves decreased steadily together with nitrate reductase and glutamine synthetase activity. Reactive oxygen species increased and oxidation-protecting compounds decreased during the life span of the leaves. Shortly after full leaf expansion an increasing number of sieve plates showed strong callose depositions when visualized by aniline blue method. At day 40 only half of the sieve tubes appeared functional. Chlorophyll breakdown followed these processes with a time lag of approximately 10 days. The sieve tube sap of ageing leaves had the same sucrose concentrations as young leaves, whereas amino acid concentrations decreased. High levels of reduced ascorbic acid and glutathione together with increasing levels of glutaredoxin indicated oxidative strain during senescence. We speculate that the gradual increase of reactive oxygen species during ageing together with the import of calcium ions lead to the stimulation of callose synthesis in plasmodesmata and sieve plates with the consequence of inhibition of phloem transport leading to carbohydrate back-up in the leaf blade. The latter may finally induce chlorophyll breakdown and, at the end, leaf abscission at the petiole base. Thus phloem blockage would precede and may be causal for chlorophyll degradation in leaf senescence.     

Chilling and forcing temperatures interact to predict the onset of wood formation in Northern Hemisphere conifers

The phenology of wood formation is a critical process to consider for predicting how trees from the temperate and boreal zones may react to climate change. Compared to leaf phenology, however, the determinism of wood phenology is still poorly known. Here, we compared for the first time three alternative ecophysiological model classes (threshold models, heat‐sum models and chilling‐influenced heat‐sum models) and an empirical model in their ability to predict the starting date of xylem cell enlargement in spring, for four major Northern Hemisphere conifers (Larix decidua, Pinus sylvestris, Picea abies and Picea mariana). We fitted models with Bayesian inference to wood phenological data collected for 220 site‐years over Europe and Canada. The chilling‐influenced heat‐sum model received most support for all the four studied species, predicting validation data with a 7.7‐day error, which is within one day of the observed data resolution. We conclude that both chilling and forcing temperatures determine the onset of wood formation in Northern Hemisphere conifers. Importantly, the chilling‐influenced heat‐sum model showed virtually no spatial bias whichever the species, despite the large environmental gradients considered. This suggests that the spring onset of wood formation is far less affected by local adaptation than by environmentally driven plasticity. In a context of climate change, we therefore expect rising winter–spring temperature to exert ambivalent effects on the spring onset of wood formation, tending to hasten it through the accumulation of forcing temperature, but imposing a higher forcing temperature requirement through the lower accumulation of chilling.     

Naturally developing memory T cell xenoreactivity to swine antigens in human peripheral blood lymphocytes

Naturally developing xenospecific Abs are well-documented barriers to xenograft transplantation in humans, but whether analogous xenoreactive T cell immunity develops is not known. We used an enzyme-linked immunospot assay to determine the frequency and cytokine profiles of xenoreactive PBLs from a panel of human volunteers. Because naive T cells produce only IL-2 in short term culture, IFN-gamma production by this approach is a measure of a memory immune response. Stimulation of human PBLs or purified T lymphocytes with stimulator cells from inbred swine revealed a high frequency of IFN-gamma producers with 5-fold fewer IL-2 producers. In contrast, lymphocytes obtained from neonatal umbilical cord blood contained swine-specific IL-2 producers but few IFN-gamma producers, which is what one would expect to find with a naive phenotype. Moreover, PBLs from adults with a history of abstention from pork consumption responded to swine cells with a significantly lower frequency of IFN-gamma producers than PBLs from adults with unrestricted diets did, suggesting that pork consumption may result in priming of swine-specific T cell immunity. Our findings provide the first evidence for naturally occurring xenospecific T cell immunity in humans. The detected strength of this memory response suggests that it will present a formidable barrier to transplantation of swine organs.     

Capacitively coupled electric fields accelerate proliferation of osteoblast-like primary cells and increase bone extracellular matrix formation in vitro

Over the last few years, electric and electromagnetic fields have gained more and more significance in the therapy of bone fracture healing and bone disease. Yet, the underlying mechanisms on a cellular and molecular level are not completely understood. In the present study we have investigated the effects of capacitively coupled, pulsed electric fields on cellular proliferation, alkaline phosphatase activity, and matrix protein synthesis of osteoblast-like primary cells in vitro. Cells were derived from bovine periosteum and electrically stimulated by saw-tooth pulses of 100 V external voltage and 16 Hz frequency. This corresponds to an electric field of 6 kV/m across the cell membranes as could be shown by computer simulation. Field application caused acceleration of cell culture development. A significant increase of proliferation concurrent with an enhancement of alkaline phosphatase activity was observed in sub-confluent cultures. Exposure of confluent osteoblast-like primary cells to electric fields resulted in enhanced synthesis and secretion of extracellular matrix-related proteins. These findings suggest that capacitively coupled electric fields accelerate bone cell proliferation and differentiation in vitro and enhance the synthesis of cells leading to promoted matrix formation and maturation.     

Endogenous cytokinin oscillations control cell cycle progression of tobacco BY-2 cells

The significance of cytokinins for the progression of the cell cycle is well known. Cytokinins contribute to the control of the expression of D-cyclins and other cell cycle genes, but knowledge as to how they affect the progression of the cell cycle is still limited. Highly synchronized tobacco BY-2 cells with clearly defined cell cycle stages were employed to determine cytokinin patterns in detail throughout the entire cycle. Concentrations of trans-zeatin, and of some other cytokinins, oscillated during the course of the cell cycle, increasing substantially at all four phase transitions and decreasing again to a minimum value during the course of each subsequent phase. Addition of exogenous cytokinins or inhibition of cytokinin biosynthesis promoted the progression of the cell cycle when the effects of these manipulations intensified the endogenous fluctuations, whereas the progression of the cycle was retarded when the amplitude of the fluctuations was decreased. The results show that the attainment of low concentrations of cytokinins is as important as the transient increases in concentration for a controlled progression from one phase of the cell cycle to the next. Cytokinin oxidase/dehydrogenase activity also showed fluctuations during the course of the cell cycle, the timing of which could at least partly explain oscillations of cytokinin levels. The activities of the enzyme were sufficient to account for the rates of cytokinin disappearance observed subsequent to a phase transition.     

Electrical stimulation influences mineral formation of osteoblast-like cells in vitro

The aim of the present study was to assess the structure of newly formed mineral crystals after electrical stimulation of osteoblast-like cells in vitro. Pulsed electrical stimulation was coupled capacitively or semi-capacitively to primary osteoblast-like cells derived from bovine metacarpals. Computer calculations revealed that the chosen input signal (saw-tooth, 100 V, 63 ms width, 16 Hz repetition rate) generated a short pulsed voltage drop of 100 microV (capacitive coupled mode) and of 350 microV (semi-capacitive coupled mode) across the cell-matrix layer. Stimulated cultures showed an enhanced mineral formation compared to the non stimulated controls. In cultures exposed to capacitively coupled electric fields and in control cultures nodules and mineralized globules were found. Nodules with a diameter of less than 200 nm covered the cell surface, whereas mineral globules with a diameter of up to 700 nm formed characteristic mineral deposits in the vicinity of the cells similar to biomineral formations occurring in mineralizing tissues. In contrast, large rod-shaped crystals were found in cultures stimulated by semi-capacitive coupled electric fields, indicating a non-physiological precipitation process. In conclusion, osteoblasts in culture are sensitive to electrical stimulation resulting in an enhancement of the biomineralization process.