Oxygen availability regulates metabolism and gene expression in trout hepatocyte cultures

We studied the metabolic rate, cellular energetic state, hypoxia-inducible factor-1 (HIF-1) activation, and expression of enzymes involved in energy metabolism using rainbow trout (Oncorhynchus mykiss) hepatocytes over the oxygen range from 21 to 1 kPa. Oxygen dependence of these factors was assessed by gradually reducing oxygen supply to cells from 21 kPa to 10, 5, 2, and 1 kPa. Moreover, time course experiments for up to 20 h at oxygen tensions of 1 and 2 kPa were carried out. Reduction of oxygen from 21 kPa to 10, 5, 2, and 1 kPa decreased metabolic rate of the cells by 14, 24, 37, and 46%, respectively. This response was instantaneous and fully reversible upon reoxygenation. Cellular ATP content and the expression of all mRNAs studied decreased when oxygen was reduced from 21 to 5 and 2 kPa. The lowest ATP levels, approximately 43% of the initial value, were measured at 5 kPa of oxygen, whereas the reduction in mRNA amounts was most pronounced at 2 kPa. At 1 kPa oxygen tension, both ATP content and mRNA amounts returned to normoxic (21 kPa) levels with a concomitant activation of HIF-1, indicating reorganization of energy metabolism in adaptation of cells to low oxygen supply. These results show that oxygen has a direct regulatory effect on metabolism of trout hepatocyte cultures, supporting the view that oxygen has a profound role in metabolic regulation in cells.     

Comparison of different normalised LCIA results and their feasibility in communication

Purpose

The purpose of this study was to answer the following three questions: (1) What are the reference values of normalisation for Finnish production and Finnish consumption and how do they differ from the European reference values?, (2) How do these differences influence the interpretation of normalised LCIA results?, and (3) How can normalised LCIA results be made more comprehensible to non-LCA experts with the help of communication material?

Methods

Finnish reference values for normalisation were calculated on the basis of the Finnish environmentally extended input–output model and ReCiPe LCIA method. The influence of different normalised results on the interpretation of LCIA was assessed based on an LCA study of print products. LCA communication material (product-specific fact sheets) was developed by organising workshops and interviews with stakeholders in the paper and printing industry.

Results and discussion

A comparison of the production based Finnish reference values to the European reference values shows that Finland contributes roughly 1 % to the European values in all impact categories except in the fossil depletion category where the contribution is 3 %. The order of magnitude of the impact categories varies depending on the reference system used for normalisation, which influences the interpretation of LCIA results. The normalised results were made more comprehensible by developing fact sheets including background information and guidance for interpretation of the LCIA results.

Conclusions

The interpreter of normalised LCIA results does not usually have the information to estimate how the chosen reference system influences the results. A sensitivity analysis with different reference values may help to highlight this effect. When communicating to non-LCA-practitioners, LCIA results need to be connected to a wider context, which can be achieved by using normalisation to give an idea of the order of magnitude of the results. However, the harmfulness of the impact categories in relation to each other cannot be judged on the basis of the normalised results, which seems to be a difficult concept for non-LCA-practitioners to understand.     

Genetic and Environmental Controls on Nitrous Oxide Accumulation in Lakes

We studied potential links between environmental factors, nitrous oxide (N₂O) accumulation, and genetic indicators of nitrite and N₂O reducing bacteria in 12 boreal lakes. Denitrifying bacteria were investigated by quantifying genes encoding nitrite and N₂O reductases (nirS/nirK and nosZ, respectively, including the two phylogenetically distinct clades nosZ _I and nosZ _II) in lake sediments. Summertime N₂O accumulation and hypolimnetic nitrate concentrations were positively correlated both at the inter-lake scale and within a depth transect of an individual lake (Lake Vanajavesi). The variability in the individual nirS, nirK, nosZ _I, and nosZ _II gene abundances was high (up to tenfold) among the lakes, which allowed us to study the expected links between the ecosystem’s nir-vs-nos gene inventories and N₂O accumulation. Inter-lake variation in N₂O accumulation was indeed connected to the relative abundance of nitrite versus N₂O reductase genes, i.e. the (nirS+nirK)/nosZ _I gene ratio. In addition, the ratios of (nirS+nirK)/nosZ _I at the inter-lake scale and (nirS+nirK)/nosZ _I+II within Lake Vanajavesi correlated positively with nitrate availability. The results suggest that ambient nitrate concentration can be an important modulator of the N₂O accumulation in lake ecosystems, either directly by increasing the overall rate of denitrification or indirectly by controlling the balance of nitrite versus N₂O reductase carrying organisms.     

Transpiration directly regulates the emissions of water‐soluble short‐chained OVOCs

Most plant‐based emissions of volatile organic compounds are considered mainly temperature dependent. However, certain oxygenated volatile organic compounds (OVOCs) have high water solubility; thus, also stomatal conductance could regulate their emissions from shoots. Due to their water solubility and sources in stem and roots, it has also been suggested that their emissions could be affected by transport in the xylem sap. Yet further understanding on the role of transport has been lacking until present. We used shoot‐scale long‐term dynamic flux data from Scots pines (Pinus sylvestris) to analyse the effects of transpiration and transport in xylem sap flow on emissions of 3 water‐soluble OVOCs: methanol, acetone, and acetaldehyde. We found a direct effect of transpiration on the shoot emissions of the 3 OVOCs. The emissions were best explained by a regression model that combined linear transpiration and exponential temperature effects. In addition, a structural equation model indicated that stomatal conductance affects emissions mainly indirectly, by regulating transpiration. A part of the temperature's effect is also indirect. The tight coupling of shoot emissions to transpiration clearly evidences that these OVOCs are transported in the xylem sap from their sources in roots and stem to leaves and to ambient air.     

Stem emissions of monoterpenes,acetaldehyde and methanol from Scots pine (Pinus sylvestris L.) affected by tree–water relations and cambial growth

Tree stems are an overlooked source of volatile organic compounds (VOCs). Their contribution to ecosystem processes and total VOC fluxes is not well studied, and assessing it requires better understanding of stem emission dynamics and their driving processes. To gain more mechanistic insight into stem emission patterns, we measured monoterpene, methanol and acetaldehyde emissions from the stems of mature Scots pines (Pinus sylvestris L.) in a boreal forest over three summers. We analysed the effects of temperature, soil water content, tree water status, transpiration and growth on the VOC emissions and used generalized linear models to test their relative importance in explaining the emissions. We show that Scots pine stems are considerable sources of monoterpenes, methanol and acetaldehyde, and their emissions are strongly regulated by temperature. However, even small changes in water availability affected the emission potentials: increased soil water content increased the monoterpene emissions within a day, whereas acetaldehyde and methanol emissions responded within 2–4 days. This lag corresponded to their transport time in the xylem sap from the roots to the stem. Moreover, the emissions of monoterpenes, methanol and acetaldehyde were influenced by the cambial growth rate of the stem with 6–10-day lags.     

The transmembrane domain is sufficient for Sbh1p function, its association with the Sec61 complex, and interaction with Rtn1p

The Sec61 protein translocation complex in the endoplasmic reticulum (ER) membrane is composed of three subunits. The alpha-subunit, called Sec61p in yeast, is a multispanning membrane protein that forms the protein conducting channel. The functions of the smaller, carboxyl-terminally tail-anchored beta subunit Sbh1p, its close homologue Sbh2p, and the gamma subunit Sss1p are not well understood. Here we show that co-translational protein translocation into the ER is reduced in sbh1Delta sbh2Delta cells, whereas there is a limited reduction of post-translational translocation and no effect on export of a mutant form of alpha-factor precursor for ER-associated degradation in the cytosol. The translocation defect and the temperature-sensitive growth phenotype of sbh1Delta sbh2Delta cells were rescued by expression of the transmembrane domain of Sbh1p alone, and the Sbh1p transmembrane domain was sufficient for coimmunoprecipitation with Sec61p and Sss1p. Furthermore, we show that Sbh1p co-precipitates with the ER transmembrane protein Rtn1p. Sbh1p-Rtn1p complexes do not appear to contain Sss1p and Sec61p. Our results define the transmembrane domain as the minimal functional domain of the Sec61beta homologue Sbh1p in ER translocation, identify a novel interaction partner for Shb1p, and imply that Sbh1p has additional functions that are not directly linked to protein translocation in association with the Sec61 complex.     

<Emphasis Type="Italic">Methylophaga</Emphasis> and <Emphasis Type="Italic">Hyphomicrobium</Emphasis> can be used as target genera in monitoring saline water methanol-utilizing denitrification

Which bacterial taxonomic groups can be used in monitoring saline water methanol-utilizing denitrification and whether nitrate is transformed into N₂ in the process are unclear. Therefore, methylotrophic bacterial communities of two efficiently functioning (nitrate/nitrite reduction was 63–96 %) tropical and cool seawater reactors at a public aquarium were investigated with clone library analysis and 454 pyrosequencing of the 16S rRNA genes. Transformation of nitrate into N₂ was confirmed using ¹⁵N labeling in incubation of carrier material from the tropical reactor. Combining the data with previous study results, Methylophaga and Hyphomicrobium were determined to be suitable target genera for monitoring the function of saline water methanol-fed denitrification systems. However, monitoring was not possible at the single species level. Interestingly, potential nitrate-reducing methylotrophs within Filomicrobium and closely related Fil I and Fil II clusters were detected in the reactors suggesting that they also contributed to methylotrophic denitrification in the saline environment.     

STR/ort mice, a model for spontaneous osteoarthritis, exhibit elevated levels of both local and systemic inflammatory markers

Osteoarthritis is a common joint disease that currently lacks disease-modifying treatments. Development of therapeutic agents for osteoarthritis requires better understanding of the disease and cost-effective in vivo models that mimic the human disease. Here, we analyzed the joints of STR/ort mice, a model for spontaneous osteoarthritis, for levels of inflammatory and oxidative stress markers and measured serum cytokines to characterize the local and systemic inflammatory status of these mice. Markers of low-grade inflammatory and oxidative stress-RAGE, AGE, S100A4, and HMGB1-were evaluated through immunohistochemistry. Of these, AGE and HMGB1 levels were elevated strongly in hyperplastic synovium, cartilage, meniscus, and ligaments in the joints of STR/ort mice compared with CBA mice, an osteoarthritis-resistant mouse strain. These increases (particularly in the synovium, meniscus, and ligaments) correlated with increased histopathologic changes in the cartilage. Serum analysis showed higher concentrations of several cytokines including IL1β, IL12p70, MIP1β, and IL5 in STR/ort mice, and these changes correlated with worsened joint morphology. These results indicate that STR/ort mice exhibited local and systemic proinflammatory conditions, both of which are present in human osteoarthritis. Therefore, the STR/ort mouse model appears to be a clinically relevant and cost-effective small animal model for testing osteoarthritis therapeutics.     

Variation in the polysome assembly and incorporation of [3H]-uridine in the cells of pine buds during the cold season

The ribosome assemblies isolated from buds of Scots pine ( Pinus sylvestris L.) containing microsporangiate strobili varied both quantitatively and qualitatively in samples collected from October to April. The seasonal fluctuation in the amount of ribosonnes was more evident in the cytosolic fraction than in the smaller membrane-bound fraction. The profiles obtained after sucrose density gradient centrifugation were of two types. One type was commonly obtained from samples collected late in the autumn and early in the spring, and this type was characterized by a relatively high peak for the large subunits, a low or negligible peak for the dimers, and an even or ascending series of peaks for the polymers. The other type was obtained from samples collected during the winter, and was characterized by small peaks for both subunits, a moderate to large peak for the dimers and a descending series of peaks for the polymers. However, the scanning electron microscope investigations indicated that the winter-time samples did not lack polysomes and clusters of ribosomes. They did not become visible in the polysome profiles because they pelleted too tightly at the bottom of the centrifuge tubes to be removed with gradient fractionation. The au-toradiographic analyses suggested that the cells were capable of synthesizing mRNA throughout the winter, whereas rRNA synthesis was arrested. On the basis of the above results, we postulate that the synthesis of the enzyme proteins needed for the maintenance of winter-time metabolism takes place in the cytosolic ribosome fraction. The possible existence of winter-time polysome stores is also pointed out.     

Dependence of the quality and the in vitro translation capacity of pine ribosome assemblies on the isolation procedure

The in vitro translation capacity of total ribosome assemblies isolated from the vegetative buds of small Scots pine (Pinus sylvestris L.) plants depends on the isolation procedure. Good yields and high values for protein synthesis were obtained in experiments in which polyvinyl pyrrolidone (PVP) was added to the grinding buffer. The polysome profiles obtained after sucrose density gradient centrifugation indicated the presence of polysomes in all samples. In addition, large ribosome aggregates were visible in the scanning electron micrographs. The use of an RNase inhibitor (RNasin) together with PVP did not improve the results, and treatment with ribonuclease (RNase, EC 3.1.27.5) destroyed the ability to synthesize protein. D, L-Dithiothreitol (DTT) and mercaptoethanol, if used instead of or together with PVP, gave low yields and also DTT destroyed the in vitro translation capacity of the ribosome assemblies. The polysome profiles had a high peak indicating dimers and often a descending series of peaks indicating polymers. A study of the scanning electron micrographs gave the impression that the largest polymers and aggregates had broken down. Protease K (EC 3.4.21.14) when added to the grinding buffer also destroyed the ability of the ribosomes to maintain protein synthesis in vitro. In this case, the shape of the polysome profiles gave the impression of successful isolation. Clumps of ribosomes, presumably originating from large aggregates, were visible in the scanning electron micrographs. Triton X-100 and 0.25 M NaCl in the grinding buffer extracted chromatin, which affected the results. The material lost during the extraction and purification processes consisted mainly of monosomes and their sub-units. On the basis of the above results it was concluded that the preservation of large polysomes and ribosome aggregates in the isolated ribosome assemblies is necessary if they are to maintain a high translation capacity. The content of the assemblies was best revealed in the scanning electron micrographs. The shape of the polysome profiles did not always correlate with the ability of the isolated ribosomes to synthesize proteins.