Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins

Summary In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.     

Mice lacking Alkbh1 display sex-ratio distortion and unilateral eye defects

Background

Eschericia coli AlkB is a 2-oxoglutarate- and iron-dependent dioxygenase that reverses alkylated DNA damage by oxidative demethylation. Mouse AlkB homolog 1 (Alkbh1) is one of eight members of the newly discovered family of mammalian dioxygenases.

Methods and Findings

In the present study we show non-Mendelian inheritance of the Alkbh1 targeted allele in mice. Both Alkbh1^−/− and heterozygous Alkbh1^+/− offspring are born at a greatly reduced frequency. Additionally, the sex-ratio is considerably skewed against female offspring, with one female born for every three to four males. Most mechanisms that cause segregation distortion, act in the male gametes and affect male fertility. The skewing of the sexes appears to be of paternal origin, and might be set in the pachythene stage of meiosis during spermatogenesis, in which Alkbh1 is upregulated more than 10-fold. In testes, apoptotic spermatids were revealed in 5–10% of the tubules in Alkbh1^−/− adults. The deficiency of Alkbh1 also causes misexpression of Bmp2, 4 and 7 at E11.5 during embryonic development. This is consistent with the incompletely penetrant phenotypes observed, particularly recurrent unilateral eye defects and craniofacial malformations.

Conclusions

Genetic and phenotypic assessment suggests that Alkbh1 mediates gene regulation in spermatogenesis, and that Alkbh1 is essential for normal sex-ratio distribution and embryonic development in mice.     

The development of an innervated epithelial barrier model using a human corneal cell line and ND7/23 sensory neurons

The corneal epithelium is a highly innervated tissue and hence in vitro models that mimic the effects of chemicals or radiation (e.g. ultra violet) on this important barrier should include consideration of the potential role of innervation. A sensory neural cell line, ND7/23, was incorporated into a 2D and 3D model of a corneal epithelium, using a human corneal cell line, and effects on barrier integrity were neither adverse nor stimulatory. In the 3D model the nerve cell bodies were separated from the corneal epithelium, via a porous polycarbonate insert membrane. The ND7/23 cells were induced to form neurites and cease division when cultured in the keratinocyte medium employed for the corneal cells. In the absence of calcium, the epithelial barrier function was lost, shown by enhanced fluorescein leakage and relocation of ZO-1 and E-cadherin from the cell membrane. At 60 microM calcium, and above, the corneal cells formed tight junctions, with peripheral membrane location of ZO-1 and E-cadherin. The presence of the ND7/23 cells did not compromise or enhance the time taken to form these junctions, when monitored at 24-h intervals over 72 h. Both male- and female-derived human corneal cell lines showed a similar tight junction functional response to different medium calcium concentrations in the presence or absence of the ND7/23 cells. Once differentiated in keratinocyte medium, patch-clamped ND7/23 cells were capable of producing a whole-cell current when exposed to low pH (5.4), indicative of the presence of active pH-gated ion channels.     

The effect of lactic acid on anaerobic carbon or nitrogen limited chemostat cultures of Saccharomyces cerevisiae

Weak organic acids are well-known metabolic effectors in yeast and other micro-organisms. High concentrations of lactic acid due to infection of lactic acid bacteria often occurs in combination with growth under nutrient-limiting conditions in industrial yeast fermentations. The effects of lactic acid on growth and product formation of Saccharomyces cerevisiae were studied, with cells growing under carbon- or nitrogen-limiting conditions in anaerobic chemostat cultures (D=0.1 h⁻¹) at pH values 3.25 and 5. It was shown that lactic acid in industrially relevant concentrations had a rather limited effect on the metabolism of S. cerevisiae. However, there was an effect on the energetic status of the cells, i.e. lactic acid addition provoked a reduction in the adenosine triphosphate (ATP) content of the cells. The decrease in ATP was not accompanied by a significant increase in the adenosine monophosphate levels.     

Stress coping style predicts aggression and social dominance in rainbow trout

Social stress is frequently used as a model for studying the neuroendocrine mechanisms underlying stress-induced behavioral inhibition, depression, and fear conditioning. It has previously been shown that social subordination may result in increased glucocorticoid release and changes in brain signaling systems. However, it is still an open question which neuroendocrine and behavioral differences are causes, and which are consequences of social status. Using juvenile rainbow trout of similar size and with no apparent differences in social history, we demonstrate that the ability to win fights for social dominance can be predicted from the duration of a behavioral response to stress, in this case appetite inhibition after transfer to a new environment. Moreover, stress responsiveness in terms of confinement-induced changes in plasma cortisol was negatively correlated to aggressive behavior. Fish that exhibited lower cortisol responses to a standardized confinement test were markedly more aggressive when being placed in a dominant social position later in the study. These findings support the view that distinct behavioral-physiological stress coping styles are present in teleost fish, and these coping characteristics influence both social rank and levels of aggression.     

Life cycle assessment of lightweight and end-of-life scenarios for generic compact class passenger vehicles

Goal, Scope and Background  The automotive industry has a long history in improving the environmental performance of vehicles - fuel economy and emission improvements, introduction of recycled and renewable materials, etc. The European Union also aims at improving the environmental performance of products by reducing, in particular, waste resulting from End-of-Life Vehicles (ELVs) for example. The European Commission estimates that ELVs contribute to approximately 1 % of the total waste in Europe [9]. Other European Union strategies are considering more life cycle aspects, as well as other impacts including resource or climate change. This article is summarizing the results of a European Commission funded project (LIRECAR) that aims at identifying the environmental impacts and relevance for combinations of recycling / recovery and lightweight vehicle design options over the whole life cycle of a vehicle - i.e. manufacturing, use and recycling/recovery. Three, independent and scientific LCA experts reviewed the study according to ISO 14040. From the beginning, representatives of all Life Cycle Stakeholders have been involved (European materials & supplier associations, an environmental Non-Governmental Organization, recycler’s association). Model and System Definition  The study compared 3 sets of theoretical vehicle weight scenarios: 1000 kg reference (material range of today’s end-of-life, mid-sized vehicles produced in the early 1990’s) and 2 lightweight scenarios for 100 kg and 250 kg less weight based on reference functions (in terms of comfort, safety, etc.) and a vehicle concept. The scenarios are represented by their material range of a broad range of lightweight strategies of most European car manufacturers. In parallel, three End-of-Life (EOL) scenarios are considered: EOL today and two theoretical extreme scenarios (100% recycling, respectively, 100% recovery of shredder residue fractions that are disposed of today). The technical and economical feasibility of the studied scenarios is not taken into consideration (e.g. 100% recycling is not possible). Results and Discussion  Significant differences between the various, studied weight scenarios were determined in several scenarios for the environmental categories of global warming, ozone depletion, photochemical oxidant creation (summer smog), abiotic resource depletion, and hazardous waste. However, these improvement potentials can be only realized under well defined conditions (e.g. material compositions, specific fuel reduction values and EOL credits) based on case-by-case assessments for improvements over the course of the life cycle. Looking at the studied scenarios, the relative contribution of the EOL phase represents 5% or less of the total life cycle impact for most selected impact categories and scenarios. The EOL technology variations studied do not impact significantly the considered environmental impacts. Exceptions include total waste, as long as stockpile goods (overburden, tailings and ore/coal processing residues) and EOL credits are considered. Conclusions and Recommendations  LIRECAR focuses only on lightweight/recycling, questions whereas other measures (changes in safety or comfort standards, propulsion improvements for CO₂, user behavior) are beyond the scope of the study. The conclusions are also not necessarily transferable to other vehicle concepts. However, for the question of end-of-life options, it can be concluded that LIRECAR cannot support any general recommendation and/or mandatory actions to improve recycling if lightweight is affected. Also, looking at each vehicle, no justification could be found for the general assumption that lightweight and recycling greatly influence the affected environmental dimension (Global Warming Potential or resource depletion and waste, respectively). LIRECAR showed that this general assumption is not true under all analyzed circumstances and not as significant as suggested. Further discussions and product development targets shall not focus on generic targets that define the approach/technology concerned with how to achieve environmental improvement (weight reduction [kg], recycling quota [%]), but on overall life cycle improvement). To enable this case-by-case assessment, exchanges of necessary information with suppliers are especially relevant.