Time‐dependent climate impact of a bioenergy system – methodology development and application to Swedish conditions

The area of dedicated energy crops is expected to increase in Sweden. This will result in direct land use changes, which may affect the carbon stocks in soil and biomass, as well as yield levels and the use of inputs. Carbon dioxide (CO₂) fluxes of biomass are often not considered when calculating the climate impact in life cycle assessments (LCA) assuming that the CO₂ released at combustion has recently been captured by the biomass in question. With the extended time lag between capture and release of CO₂ inherent in many perennial bioenergy systems, the relation between carbon neutrality and climate neutrality may be questioned. In this paper, previously published methodologies and models are combined in a methodological framework that can assist LCA practitioners in interpreting the time‐dependent climate impact of a bioenergy system. The treatment of carbon differs from conventional LCA practice in that no distinction is made between fossil and biogenic carbon. A time‐dependent indicator is used to enable a representation of the climate impact that is not dependent on the choice of a specific characterization time horizon or time of evaluation and that does not use characterization factors, such as global warming potential and global temperature potential. The indicator used to aid in the interpretation phase of this paper is global mean surface temperature change (ΔT_s(n)). A theoretical system producing willow for district heating was used to study land use change effects depending on previous land use and variations in the standing biomass carbon stocks. When replacing annual crops with willow this system presented a cooling contribution to ΔT_s(n). However, the first years after establishing the willow plantation it presented a warming contribution to ΔT_s(n). This behavior was due mainly to soil organic carbon (SOC) variation. A rapid initial increase in standing biomass counteracted the initial SOC loss.     

Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry

The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time -a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift -was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonie medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 μM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.     

25-Hydroxylation of vitamin D3 by a reconstituted system from rat liver microsomes.

Using isotope dilution—mass fragmentography as assay technique, it was shown that highly purified preparations of cytochrome P-450 from rat liver microsomes catalyzed 25-hydroxylation of vitamin D₃ when combined with NADPH-cytochrome P-450 reductase and a phospholipid. The rate of conversion was approximately linear with the amount of cytochrome P-450, and was considerably higher than the rate of conversion obtained with crude liver microsomes. The possibility is discussed that the microsomal fraction contains inhibitors of 25-hydroxylase activity, which may be of regulatory importance in vitamin D₃ metabolism.     

Assay of liver microsomal cholesterol 7 alpha-hydroxylase using deuterated carrier and gas chromatography-mass spectrometry

The activity of cholesterol 7α-hydroxylase in rat liver microsomes was assayed by measuring the mass of 5-cholestene-3β, 7α-diol formed from endogenous cholesterol under standardized incubation conditions. After termination of incubations, a known amount of 5-[24,25,7β-²H₃]cholestene-3β,7α-diol was added. A chloroform extract of the incubation mixture was subjected to thin layer chromatography and the fraction containing 5-cholestene-3β,7α-diol was converted into trimethylsilyl ether. The trimethylsilyl ether was subjected to combined gas chromatography-mass spectrometry and the amount of unlabeled 5-cholestene-3β,7α-diol in the mixture was calculated from the ratio between the relative intensitics of the peaks at $\text{m}\text{e}$ 456 (M-90) and $\text{m}\text{e}$ 459 [M-(90 + 3)]. The precision of the method was ±2.2% (SD). The results with this method of assay of cholesterol 7α-hydroxylase were compared with those obtained with a method based on conversion of a trace amount of added [4-¹⁴C]cholesterol into 5-cholestene-3β,7α-diol.     

FATTY ACID CONTENT AND CHEMICAL COMPOSITION OF FRESHWATER MICROALGAE1

Fatty acid (FA), total lipid, protein, amino acid, carbon, nitrogen, and phosphorus content was analyzed in 24 samples of freshwater microalgae. The samples originated from batch, continuous, or mass cultures in various growth phases and from net samples from lakewater. FA were analyzed quantitatively by using an internal standard in a GLC system and expressed as mg·g^?1 dry weight (DW). The FA of one group of blue-greens (e.g. Oscillatoria and Microcystis) were similar to those of the greens with higher amounts of 18C acids of the ω3 type compared to the ω6 type, whereas the other group (e.g. Anabaena and Spirulina) contained mostly ω6 acids. The flagellates, a taxonomically diverse group, were characterized by high amounts of long-chained (20–22 C) polyunsaturated FA (PUFA), particularly of the ω3 type. The ω3/ω6 ratio appears to be highest in algae in the exponential growth phase. The increased lipid content in stressed algae was mostly due to increased saturated fatty acids and ω6 acids, whereas the valuable ω3 acids were unchanged or even decreased. Amino acid composition (% of total amino acids) did not vary much betaken species, but when analyzed quantitatively (mg-g^?1 DW), varied considerably between species and within species in different growth phases. The nitrogen and phosphorus contents were variable in all three algal groups. The relationship between PUFA and phosphorus content differed among the algal groups. The data suggest that PUFA in the phospholipids consist mostly ω3 acids.     

Marked change in the balance between CYP27A1 and CYP46A1 mediated elimination of cholesterol during differentiation of human neuronal cells

Cholesterol metabolism in the brain is distinct from that in other tissues due to the fact that cholesterol itself is unable to pass across the blood-brain barrier. Elimination of brain cholesterol is mainly dependent on a neuronal-specific cytochrome P450, CYP46A1, catalyzing the conversion of cholesterol into 24(S)-hydroxycholesterol (24OHC), which is able to pass the blood-brain barrier. A suitable model for studying this elimination from human neuronal cells has not been described previously. It is shown here that differentiated Ntera2/clone D1 (NT2) cells express the key genes involved in brain cholesterol homeostasis including CYP46A1, and that the expression profiles of the genes observed during neuronal differentiation are those expected to occur in vivo. Thus there was a decrease in the mRNA levels corresponding to cholesterol synthesis enzymes and a marked increase in the mRNA level of CYP46A1. The latter increase was associated with increased levels of CYP46A1 protein and increased production of 24OHC. The magnitude of the secretion of 24OHC from the differentiated NT2 cells into the medium was similar to that expected to occur under in vivo conditions. An alternative to elimination of cholesterol by the CYP46A1 mechanism is elimination by CYP27A1, and the product of this enzyme, 27-hydroxycholesterol (27OHC), is also known to pass the blood-brain barrier. The CYP27A1 protein level decreased during the differentiation of the NT2 cells in parallel with decreased production of 27OHC. The ratio between 24OHC and 27OHC in the medium from the cultured cells increased, by a factor of 13, during the differentiation process. The results suggest that progenitor cells eliminate cholesterol in the form of 27OHC while neurogenesis induces a change to the CYP46A1 dependent pathway. Furthermore this study demonstrates that differentiated NT2 cells are suitable for studies of cholesterol homeostasis in human neurons.     

Characterization of Cholesterol Homeostasis in Telomerase-immortalized Tangier Disease Fibroblasts Reveals Marked Phenotype Variability

We compared the consequences of an ABCA1 mutation that produced an apparent lack of atherosclerosis (Tangier family 1, N935S) with an ABCA1 mutation with functional ABCA1 knockout that was associated with severe atherosclerosis (Tangier family 2, Leu⁵⁴⁸:Leu⁵⁷⁵-End), using primary and telomerase-immortalized fibroblasts. Telomerase-immortalized Tangier fibroblasts of family 1 (TT1) showed 30% residual cholesterol efflux capacity in response to apolipoprotein A-I, whereas telomerase-immortalized Tangier fibroblasts of family 2 (TT2) showed only 20%. However, there were a number of secondary differences that were often stronger and may help to explain the more rapid development of atherosclerosis in family 2. First, the total cellular cholesterol content increase was 2–3-fold and 3–5-fold in TT1 and TT2 cells, respectively. The corresponding increase in esterified cholesterol concentration was 10- and 40-fold, respectively. Second, 24-, 25-, and 27-hydroxycholesterol concentrations were moderately increased in TT1 cells, but were increased as much as 200-fold in TT2 cells. Third, cholesterol biosynthesis was moderately decreased in TT1 cells, but was markedly decreased in TT2 cells. Fourth, potentially atheroprotective LXR-dependent SREBP1c signaling was normal in TT1, but was rather suppressed in TT2 cells. Cultivated primary Tangier fibroblasts were characterized by premature aging in culture and were associated with less obvious biochemical differences. In summary, these results may help to understand the differential atherosclerotic susceptibility in Tangier disease and further demonstrate the usefulness of telomerase-immortalized cells in studying this cellular phenotype. The data support the contention that side chain-oxidized oxysterols are strong suppressors of cholesterol biosynthesis under specific pathological conditions in humans.     

Importance of lysine 125 for heparin binding and activation of antithrombin

The anticoagulant sulfated polysaccharide, heparin, binds to the plasma coagulation proteinase inhibitor, antithrombin, and activates it by a conformational change that results in a greatly increased rate of inhibition of target proteinases. Lys125 of antithrombin has previously been implicated in this binding by chemical modification and site-directed mutagenesis and by the crystal structure of a complex between antithrombin and a pentasaccharide constituting the antithrombin-binding region of heparin. Replacement of Lys125 with Met or Gln in this work reduced the affinity of antithrombin for full-length heparin or the pentasaccharide by 150-600-fold at I = 0.15, corresponding to a loss of 25-33% of the total binding energy. The affinity decrease was due both to disruption of approximately three ionic interactions, indicating that Lys125 and two other basic residues of antithrombin act cooperatively in binding to heparin, and to weakened nonionic interactions. The mutations caused a 10-17-fold decrease in the affinity of the initial, weak binding step of the two-step mechanism of heparin binding to antithrombin. They also increased the reverse rate constant of the second, conformational change step by 10-50-fold. Lys125 is thus a major heparin-binding residue of antithrombin, contributing an amount of binding energy comparable to that of Arg129, but less energy than Lys114. It is the first residue identified so far that has a critical role in the initial recognition of heparin by antithrombin, but also appreciably stabilizes the heparin-induced activated state of the inhibitor. These effects are exerted by interactions of Lys125 with the nonreducing end of the heparin pentasaccharide.     

Symbionts as major modulators of insect health: lactic acid bacteria and honeybees

Lactic acid bacteria (LAB) are well recognized beneficial host-associated members of the microbiota of humans and animals. Yet LAB-associations of invertebrates have been poorly characterized and their functions remain obscure. Here we show that honeybees possess an abundant, diverse and ancient LAB microbiota in their honey crop with beneficial effects for bee health, defending them against microbial threats. Our studies of LAB in all extant honeybee species plus related apid bees reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect and suggest a long (>80 mya) history of association. Bee associated microbiotas highlight Lactobacillus kunkeei as the dominant LAB member. Those showing potent antimicrobial properties are acquired by callow honey bee workers from nestmates and maintained within the crop in biofilms, though beekeeping management practices can negatively impact this microbiota. Prophylactic practices that enhance LAB, or supplementary feeding of LAB, may serve in integrated approaches to sustainable pollinator service provision. We anticipate this microbiota will become central to studies on honeybee health, including colony collapse disorder, and act as an exemplar case of insect-microbe symbiosis.     

The structure of the desiccated <Emphasis Type="Italic">Richtersius coronifer</Emphasis> (Richters, 1903)

Tun formation is an essential morphological adaptation for entering the anhydrobiotic state in tardigrades, but its internal structure has rarely been investigated. We present the structure and ultrastructure of organs and cells in desiccated Richtersius coronifer by transmission and scanning electron microscopy, confocal microscopy, and histochemical methods. A 3D reconstruction of the body organization of the tun stage is also presented. The tun formation during anhydrobiosis of tardigrades is a process of anterior-posterior body contraction, which relocates some organs such as the pharyngeal bulb. The cuticle is composed of epicuticle, intracuticle and procuticle; flocculent coat; and trilaminate layer. Moulting does not seem to restrict the tun formation, as evidenced from tardigrade tuns that were in the process of moulting. The storage cells of desiccated specimens filled up the free inner space and surrounded internal organs, such as the ovary and digestive system, which were contracted. All cells (epidermal cells, storage cells, ovary cells, cells of the digestive system) underwent shrinkage, and their cytoplasm was electron dense. Lipids and polysaccharides dominated among reserve material of storage cells, while the amount of protein was small. The basic morphology of specific cell types and organelles did not differ between active and anhydrobiotic R. coronifer.